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Zhou, Pei

Overview:

Protein-protein interactions play a pivotal role in the regulation of various cellular processes. The formation of higher order protein complexes is frequently accompanied by extensive structural remodeling of the individual components, varying from domain re-orientation to induced folding of unstructured elements. Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for macromolecular structure determination in solution. It has the unique advantage of being capable of elucidating the dynamic behavior of proteins during the process of recognition. Recent advances in NMR techniques have enabled the study of significantly larger proteins and protein complexes. These innovations have also led to faster and more accurate structure determination. My research interests focus on the exploration of molecular recognition and conformation variability of protein complexes in crucial biomedical processes using state-of-the-art NMR techniques.

Positions:

Professor of Biochemistry

Biochemistry
School of Medicine

Professor of Chemistry

Chemistry
Trinity College of Arts & Sciences

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1998

Ph.D. — Harvard University

Post Doct Fellow, Biological Chemistry

Harvard University

Grants:

Deep Topological Sampling of Protein Structures

Administered By
Computer Science
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
September 18, 2017
End Date
July 31, 2021

Structure-Function Analysis of Chlamydia Secretion Chaperones

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
February 01, 2016
End Date
January 31, 2021

Biochemistry and Structure of Lipid A Enzymes

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
March 01, 2016
End Date
February 29, 2020

Molecular targets in peptidoglycan synthesis

Administered By
Biochemistry
AwardedBy
Medical University of South Carolina
Role
Principal Investigator
Start Date
February 01, 2016
End Date
January 31, 2018

Targeting Rev1-mediated Translesion Synthesis for Cancer Therapy

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 2015
End Date
June 30, 2017

LpxC inhibitors as a novel class of antibiotics against N. gonorrhoeae

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
April 01, 2011
End Date
March 31, 2017

Preliminary Safety Screening of Duke PlxC inhibitors as Novel Antibiotics against Gram-negative Pathogens

Administered By
Biochemistry
AwardedBy
North Carolina Biotechnology Center
Role
Principal Investigator
Start Date
November 01, 2015
End Date
October 31, 2016

Structural Biology and Biophysics Training Program

Administered By
Basic Science Departments
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1994
End Date
September 30, 2015

Biosynthesis And Function Of Lipopolysaccharides

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 01, 1994
End Date
July 31, 2015

High sensitivity multi-purpose electron paramagnetic resonance spectroscopy for biotechnological and biomedical research

Administered By
Biochemistry
AwardedBy
North Carolina Biotechnology Center
Role
Collaborating Investigator
Start Date
May 01, 2014
End Date
April 30, 2015

Automated detection of protein crystals in high-throughput crystallography experiments

Administered By
Duke Human Vaccine Institute
AwardedBy
North Carolina Biotechnology Center
Role
Major User
Start Date
April 01, 2014
End Date
April 30, 2015

New Console and Cold Probe for the Duke 600 MHz NMR Spectrometer System

Administered By
Radiology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
June 15, 2013
End Date
June 14, 2014

Structural and Biochemical Studies of LpxC Inhibition

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 15, 2003
End Date
March 31, 2014

Lipid A Modification Systems in Gram-Negative Bacteria

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1995
End Date
October 31, 2013

Automated NMR Assignment and Protein Structure Determination

Administered By
Computer Science
AwardedBy
National Institutes of Health
Role
Co-Principal Investigator
Start Date
September 16, 2006
End Date
November 30, 2012

Structural, Biochemical and Functional Studies of RNAPII CTD Interacting Proteins

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2008
End Date
July 31, 2012

Replacement Equipment Components for an 800 MHz NMR Spectrometer

Administered By
Radiology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
August 20, 2010
End Date
August 19, 2011

Regulation of Germline Stem Cell Division in Drosophila

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
February 01, 1996
End Date
June 07, 2006

Structure and Regulation of hsp90 Chaperones

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
May 01, 2005
End Date
October 31, 2005
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Publications:

Structure, inhibition, and regulation of essential lipid A enzymes.

The Raetz pathway of lipid A biosynthesis plays a vital role in the survival and fitness of Gram-negative bacteria. Research efforts in the past three decades have identified individual enzymes of the pathway and have provided a mechanistic understanding of the action and regulation of these enzymes at the molecular level. This article reviews the discovery, biochemical and structural characterization, and regulation of the essential lipid A enzymes, as well as continued efforts to develop novel antibiotics against Gram-negative pathogens by targeting lipid A biosynthesis. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Authors
Zhou, P; Zhao, J
MLA Citation
Zhou, P, and Zhao, J. "Structure, inhibition, and regulation of essential lipid A enzymes." Biochimica et biophysica acta 1862.11 (November 2017): 1424-1438. (Review)
Website
http://hdl.handle.net/10161/13289
PMID
27940308
Source
epmc
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1862
Issue
11
Publish Date
2017
Start Page
1424
End Page
1438
DOI
10.1016/j.bbalip.2016.11.014

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria.IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

Authors
Lemaître, N; Liang, X; Najeeb, J; Lee, C-J; Titecat, M; Leteurtre, E; Simonet, M; Toone, EJ; Zhou, P; Sebbane, F
MLA Citation
Lemaître, N, Liang, X, Najeeb, J, Lee, C-J, Titecat, M, Leteurtre, E, Simonet, M, Toone, EJ, Zhou, P, and Sebbane, F. "Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC." mBio 8.4 (July 25, 2017).
PMID
28743813
Source
epmc
Published In
mBio
Volume
8
Issue
4
Publish Date
2017
DOI
10.1128/mbio.00674-17

Probing the excited-state chemical shifts and exchange parameters by nitrogen-decoupled amide proton chemical exchange saturation transfer (HNdec-CEST).

CEST-NMR spectroscopy is a powerful tool for probing the conformational dynamics of macromolecules. We present a HNdec-CEST experiment that simplifies the relaxation matrix, reduces fitting parameters, and enhances signal resolution. Importantly, fitting of HNdec-CEST profiles enables robust extraction of exchange rates as well as excited-state chemical shifts and populations.

Authors
Wu, Q; Fenton, BA; Wojtaszek, JL; Zhou, P
MLA Citation
Wu, Q, Fenton, BA, Wojtaszek, JL, and Zhou, P. "Probing the excited-state chemical shifts and exchange parameters by nitrogen-decoupled amide proton chemical exchange saturation transfer (HNdec-CEST)." Chemical communications (Cambridge, England) 53.61 (July 14, 2017): 8541-8544.
PMID
28707688
Source
epmc
Published In
Chemical Communications
Volume
53
Issue
61
Publish Date
2017
Start Page
8541
End Page
8544
DOI
10.1039/c7cc05021f

The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA.

Plant development requires coordination among complex signaling networks to enhance the plant's adaptation to changing environments. DELLAs, transcription regulators originally identified as repressors of phytohormone gibberellin signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we found that DELLA is mono-O-fucosylated by the novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3) and PIF4. Moreover, spy mutants displayed elevated responses to gibberellin and brassinosteroid, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding may have broader importance because SPY orthologs are conserved in prokaryotes and eukaryotes, thus suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms.

Authors
Zentella, R; Sui, N; Barnhill, B; Hsieh, W-P; Hu, J; Shabanowitz, J; Boyce, M; Olszewski, NE; Zhou, P; Hunt, DF; Sun, T-P
MLA Citation
Zentella, R, Sui, N, Barnhill, B, Hsieh, W-P, Hu, J, Shabanowitz, J, Boyce, M, Olszewski, NE, Zhou, P, Hunt, DF, and Sun, T-P. "The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA." Nature chemical biology 13.5 (May 2017): 479-485.
PMID
28244988
Source
epmc
Published In
Nature Chemical Biology
Volume
13
Issue
5
Publish Date
2017
Start Page
479
End Page
485
DOI
10.1038/nchembio.2320

CLEAN

© The Royal Society of Chemistry 2017. CLEAN is an algorithm for the suppression of artifacts from nonuniform sampling, originally developed in the field of radioastronomy in the 1960s and 1970s. Recognizing similarities between the problem of NMR NUS and astronomical data collection, several NMR groups have applied versions of CLEAN to NMR data. Here, we recount the historical background of CLEAN in astronomy, explain how the algorithm works as applied to NMR, examine the method's theoretical underpinnings, and present examples of its use with NMR experiments. CLEAN shows a number of similarities to methods being used in the compressed sensing community, and recent theoretical results suggest that CLEAN should succeed under the same or similar conditions to those in which convex l 1 -norm minimization succeeds.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "CLEAN." New Developments in NMR. January 1, 2017. 169-219.
Source
scopus
Volume
2017-January
Publish Date
2017
Start Page
169
End Page
219
DOI
10.1039/9781782628361-00169

Backprojection and Related Methods

© The Royal Society of Chemistry 2017. The sampling of the NMR time domain along radial spokes allows one to obtain projections of an NMR spectrum at various angles. These projections encode information about the positions, intensities, and lineshapes of the peaks in the spectrum, and this information can be recovered using suitable reconstruction methods. Here, we describe the technique of radial data collection and outline an intuitive framework for approaching reconstruction. We then survey the theory of tomographic reconstruction, and use that theory to analyze reconstruction methods and the information available from radial sampling. Finally, we survey past applications of projection-reconstruction methodology in NMR, including its use in 3-D and 4-D NMR data including NOESY.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "Backprojection and Related Methods." New Developments in NMR. January 1, 2017. 119-168.
Source
scopus
Volume
2017-January
Publish Date
2017
Start Page
119
End Page
168
DOI
10.1039/9781782628361-00119

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics.MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively.LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with β-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group.These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

Authors
Titecat, M; Liang, X; Lee, C-J; Charlet, A; Hocquet, D; Lambert, T; Pagès, J-M; Courcol, R; Sebbane, F; Toone, EJ; Zhou, P; Lemaitre, N
MLA Citation
Titecat, M, Liang, X, Lee, C-J, Charlet, A, Hocquet, D, Lambert, T, Pagès, J-M, Courcol, R, Sebbane, F, Toone, EJ, Zhou, P, and Lemaitre, N. "High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors." The Journal of antimicrobial chemotherapy 71.10 (October 2016): 2874-2882.
Website
http://hdl.handle.net/10161/12518
PMID
27330072
Source
epmc
Published In
Journal of Antimicrobial Chemotherapy
Volume
71
Issue
10
Publish Date
2016
Start Page
2874
End Page
2882
DOI
10.1093/jac/dkw210

Structure of the essential Haemophilus influenzae UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis.

In most Gram-negative pathogens, the hydrolysis of UDP-2,3-diacylglucosamine to generate lipid X in lipid A biosynthesis is catalysed by the membrane-associated enzyme LpxH. We report the crystal structure of LpxH in complex with its product, lipid X, unveiling a unique insertion lid above the conserved architecture of calcineurin-like phosphoesterases. This structure reveals elaborate interactions surrounding lipid X and provides molecular insights into the substrate selectivity, catalysis and inhibition of LpxH.

Authors
Cho, J; Lee, C-J; Zhao, J; Young, HE; Zhou, P
MLA Citation
Cho, J, Lee, C-J, Zhao, J, Young, HE, and Zhou, P. "Structure of the essential Haemophilus influenzae UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis." Nature microbiology 1.11 (August 15, 2016): 16154-.
Website
http://hdl.handle.net/10161/13058
PMID
27780190
Source
epmc
Published In
Nature microbiology
Volume
1
Issue
11
Publish Date
2016
Start Page
16154
DOI
10.1038/nmicrobiol.2016.154

Unbiased measurements of reconstruction fidelity of sparsely sampled magnetic resonance spectra.

The application of sparse-sampling techniques to NMR data acquisition would benefit from reliable quality measurements for reconstructed spectra. We introduce a pair of noise-normalized measurements, and , for differentiating inadequate modelling from overfitting. While and can be used jointly for methods that do not enforce exact agreement between the back-calculated time domain and the original sparse data, the cross-validation measure is applicable to all reconstruction algorithms. We show that the fidelity of reconstruction is sensitive to changes in and that model overfitting results in elevated and reduced spectral quality.

Authors
Wu, Q; Coggins, BE; Zhou, P
MLA Citation
Wu, Q, Coggins, BE, and Zhou, P. "Unbiased measurements of reconstruction fidelity of sparsely sampled magnetic resonance spectra." Nature communications 7 (July 27, 2016): 12281-.
Website
http://hdl.handle.net/10161/13065
PMID
27459896
Source
epmc
Published In
Nature Communications
Volume
7
Publish Date
2016
Start Page
12281
DOI
10.1038/ncomms12281

A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058.

The difluoromethyl-allo-threonyl hydroxamate-based compound LPC-058 is a potent inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) in Gram-negative bacteria. A scalable synthesis of this compound is described. The key step in the synthetic sequence is a transition metal/base-catalyzed aldol reaction of methyl isocyanoacetate and difluoroacetone, giving rise to 4-(methoxycarbonyl)-5,5-disubstituted 2-oxazoline. A simple NMR-based determination of enantiomeric purity is also described.

Authors
Liang, X; Gopalaswamy, R; Navas, F; Toone, EJ; Zhou, P
MLA Citation
Liang, X, Gopalaswamy, R, Navas, F, Toone, EJ, and Zhou, P. "A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058." The Journal of organic chemistry 81.10 (May 2016): 4393-4398.
Website
http://hdl.handle.net/10161/12063
PMID
27128325
Source
epmc
Published In
The Journal of Organic Chemistry
Volume
81
Issue
10
Publish Date
2016
Start Page
4393
End Page
4398
DOI
10.1021/acs.joc.6b00589

Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.

Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.

Authors
Young, HE; Zhao, J; Barker, JR; Guan, Z; Valdivia, RH; Zhou, P
MLA Citation
Young, HE, Zhao, J, Barker, JR, Guan, Z, Valdivia, RH, and Zhou, P. "Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis." mBio 7.2 (March 22, 2016): e00090-.
Website
http://hdl.handle.net/10161/11783
PMID
27006461
Source
epmc
Published In
mBio
Volume
7
Issue
2
Publish Date
2016
Start Page
e00090
DOI
10.1128/mbio.00090-16

Drug design from the cryptic inhibitor envelope.

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

Authors
Lee, C-J; Liang, X; Wu, Q; Najeeb, J; Zhao, J; Gopalaswamy, R; Titecat, M; Sebbane, F; Lemaitre, N; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Wu, Q, Najeeb, J, Zhao, J, Gopalaswamy, R, Titecat, M, Sebbane, F, Lemaitre, N, Toone, EJ, and Zhou, P. "Drug design from the cryptic inhibitor envelope." Nature communications 7 (February 25, 2016): 10638-.
Website
http://hdl.handle.net/10161/11782
PMID
26912110
Source
epmc
Published In
Nature Communications
Volume
7
Publish Date
2016
Start Page
10638
DOI
10.1038/ncomms10638

A multi-frequency optoelectronic oscillator based on a single phase-modulator

© 2015 OSA. A multi-frequency optoelectronic oscillator is proposed based on a single phase modulator. Simultaneous generation of 10 and 40 GHz signals is demonstrated and the phase noise is -100.62 dBc/Hz and -84.64 dBc/Hz@ 10kHz offset, respectively.

Authors
Zhou, P; Zhang, F; Pan, S
MLA Citation
Zhou, P, Zhang, F, and Pan, S. "A multi-frequency optoelectronic oscillator based on a single phase-modulator." August 10, 2015.
Source
scopus
Published In
Conference on Lasers and Electro-Optics Europe - Technical Digest
Volume
2015-August
Publish Date
2015

Suppression of conformational heterogeneity at a protein-protein interface.

Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.

Authors
Deis, LN; Wu, Q; Wang, Y; Qi, Y; Daniels, KG; Zhou, P; Oas, TG
MLA Citation
Deis, LN, Wu, Q, Wang, Y, Qi, Y, Daniels, KG, Zhou, P, and Oas, TG. "Suppression of conformational heterogeneity at a protein-protein interface." Proceedings of the National Academy of Sciences of the United States of America 112.29 (July 8, 2015): 9028-9033.
Website
http://hdl.handle.net/10161/10595
PMID
26157136
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
112
Issue
29
Publish Date
2015
Start Page
9028
End Page
9033
DOI
10.1073/pnas.1424724112

Systematic solution to homo-oligomeric structures determined by NMR.

Protein structure determination by NMR has predominantly relied on simulated annealing-based conformational search for a converged fold using primarily distance constraints, including constraints derived from nuclear Overhauser effects, paramagnetic relaxation enhancement, and cysteine crosslinkings. Although there is no guarantee that the converged fold represents the global minimum of the conformational space, it is generally accepted that good convergence is synonymous to the global minimum. Here, we show such a criterion breaks down in the presence of large numbers of ambiguous constraints from NMR experiments on homo-oligomeric protein complexes. A systematic evaluation of the conformational solutions that satisfy the NMR constraints of a trimeric membrane protein, DAGK, reveals 9 distinct folds, including the reported NMR and crystal structures. This result highlights the fundamental limitation of global fold determination for homo-oligomeric proteins using ambiguous distance constraints and provides a systematic solution for exhaustive enumeration of all satisfying solutions.

Authors
Martin, JW; Zhou, P; Donald, BR
MLA Citation
Martin, JW, Zhou, P, and Donald, BR. "Systematic solution to homo-oligomeric structures determined by NMR." Proteins 83.4 (April 2015): 651-661.
PMID
25620116
Source
epmc
Published In
Proteins: Structure, Function and Bioinformatics
Volume
83
Issue
4
Publish Date
2015
Start Page
651
End Page
661
DOI
10.1002/prot.24768

Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain.

FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. The ubiquitin-binding capacity of the FAAP20 UBZ is required for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery. Although the UBZ-ubiquitin interaction is thought to be exclusively encapsulated within the ββα module of UBZ, we show that the FAAP20-ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended β-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44(Ub) for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20-ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.

Authors
Wojtaszek, JL; Wang, S; Kim, H; Wu, Q; D'Andrea, AD; Zhou, P
MLA Citation
Wojtaszek, JL, Wang, S, Kim, H, Wu, Q, D'Andrea, AD, and Zhou, P. "Ubiquitin recognition by FAAP20 expands the complex interface beyond the canonical UBZ domain." Nucleic Acids Research 42.22 (December 2014): 13997-14005.
Website
http://hdl.handle.net/10161/13468
PMID
25414354
Source
epmc
Published In
Nucleic Acids Research
Volume
42
Issue
22
Publish Date
2014
Start Page
13997
End Page
14005
DOI
10.1093/nar/gku1153

Structural basis of lipid binding for the membrane-embedded tetraacyldisaccharide-1-phosphate 4'-kinase LpxK.

The membrane-bound tetraacyldisaccharide-1-phosphate 4'-kinase, LpxK, catalyzes the sixth step of the lipid A (Raetz) biosynthetic pathway and is a viable antibiotic target against emerging Gram-negative pathogens. We report the crystal structure of lipid IVA, the LpxK product, bound to the enzyme, providing a rare glimpse into interfacial catalysis and the surface scanning strategy by which many poorly understood lipid modification enzymes operate. Unlike the few previously structurally characterized proteins that bind lipid A or its precursors, LpxK binds almost exclusively to the glucosamine/phosphate moieties of the lipid molecule. Steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and characterization of N-terminal helix truncation mutants uncovers the role of this substructure as a hydrophobic membrane anchor. These studies make critical contributions to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates and provide a structural template for designing small molecule inhibitors targeting this essential kinase.

Authors
Emptage, RP; Tonthat, NK; York, JD; Schumacher, MA; Zhou, P
MLA Citation
Emptage, RP, Tonthat, NK, York, JD, Schumacher, MA, and Zhou, P. "Structural basis of lipid binding for the membrane-embedded tetraacyldisaccharide-1-phosphate 4'-kinase LpxK." The Journal of biological chemistry 289.35 (August 2014): 24059-24068.
PMID
25023290
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
35
Publish Date
2014
Start Page
24059
End Page
24068
DOI
10.1074/jbc.m114.589986

A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

Authors
Sampson, TR; Napier, BA; Schroeder, MR; Louwen, R; Zhao, J; Chin, C-Y; Ratner, HK; Llewellyn, AC; Jones, CL; Laroui, H; Merlin, D; Zhou, P; Endtz, HP; Weiss, DS
MLA Citation
Sampson, TR, Napier, BA, Schroeder, MR, Louwen, R, Zhao, J, Chin, C-Y, Ratner, HK, Llewellyn, AC, Jones, CL, Laroui, H, Merlin, D, Zhou, P, Endtz, HP, and Weiss, DS. "A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion." Proceedings of the National Academy of Sciences of the United States of America 111.30 (July 14, 2014): 11163-11168.
PMID
25024199
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
111
Issue
30
Publish Date
2014
Start Page
11163
End Page
11168
DOI
10.1073/pnas.1323025111

Sparsely-sampled, high-resolution 4-D omit spectra for detection and assignment of intermolecular NOEs of protein complexes.

Unambiguous detection and assignment of intermolecular NOEs are essential for structure determination of protein complexes by NMR. Such information has traditionally been obtained with 3-D half-filtered experiments, where scalar coupling-based purging of intramolecular signals allows for selective detection of intermolecular NOEs. However, due to the large variation of (1)JHC scalar couplings and limited chemical shift dispersion in the indirect proton dimension, it is difficult to obtain reliable and complete assignments of interfacial NOEs. Here, we demonstrate a strategy that combines selective labeling and high-resolution 4-D NOE spectroscopy with sparse sampling for reliable identification and assignment of intermolecular NOEs. Spectral subtraction of component-labeled complexes from a uniformly-labeled protein complex yields an "omit" spectrum containing positive intermolecular NOEs with little signal degeneracy. Such a strategy can be broadly applied to unbiased detection, assignment and presentation of intermolecular NOEs of protein complexes.

Authors
Wang, S; Zhou, P
MLA Citation
Wang, S, and Zhou, P. "Sparsely-sampled, high-resolution 4-D omit spectra for detection and assignment of intermolecular NOEs of protein complexes." Journal of biomolecular NMR 59.2 (June 2014): 51-56.
PMID
24789524
Source
epmc
Published In
Journal of Biomolecular NMR
Volume
59
Issue
2
Publish Date
2014
Start Page
51
End Page
56
DOI
10.1007/s10858-014-9834-2

Crystal Structure of MraY, an Essential Membrane Enzyme for Bacterial Cell Wall Synthesis

Authors
Chung, BC; Zhao, J; Gillespie, R; Kwon, DY; Guan, Z; Hong, J; Zhou, P; Lee, S-Y
MLA Citation
Chung, BC, Zhao, J, Gillespie, R, Kwon, DY, Guan, Z, Hong, J, Zhou, P, and Lee, S-Y. "Crystal Structure of MraY, an Essential Membrane Enzyme for Bacterial Cell Wall Synthesis." January 28, 2014.
Source
wos-lite
Published In
Biophysical Journal
Volume
106
Issue
2
Publish Date
2014
Start Page
14A
End Page
14A

Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC.

The LpxC enzyme in the lipid A biosynthetic pathway is one of the most promising and clinically unexploited antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Progress in medicinal chemistry has led to the discovery of potent LpxC inhibitors with a variety of chemical scaffolds and distinct antibiotic profiles. The vast majority of these compounds, including the nanomolar inhibitors L-161,240 and BB-78485, are highly effective in suppressing the activity of Escherichia coli LpxC (EcLpxC) but not divergent orthologs such as Pseudomonas aeruginosa LpxC (PaLpxC) in vitro. The molecular basis for such promiscuous inhibition of EcLpxC has remained poorly understood. Here, we report the crystal structure of EcLpxC bound to L-161,240, providing the first molecular insight into L-161,240 inhibition. Additionally, structural analysis of the EcLpxC/L-161,240 complex together with the EcLpxC/BB-78485 complex reveals an unexpected backbone flipping of the Insert I βa-βb loop in EcLpxC in comparison with previously reported crystal structures of EcLpxC complexes with l-threonyl-hydroxamate-based broad-spectrum inhibitors. Such a conformational switch, which has only been observed in EcLpxC but not in divergent orthologs such as PaLpxC, results in expansion of the active site of EcLpxC, enabling it to accommodate LpxC inhibitors with a variety of head groups, including compounds containing single (R- or S-enantiomers) or double substitutions at the neighboring Cα atom of the hydroxamate warhead group. These results highlight the importance of understanding inherent conformational plasticity of target proteins in lead optimization.

Authors
Lee, C-J; Liang, X; Gopalaswamy, R; Najeeb, J; Ark, ED; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Gopalaswamy, R, Najeeb, J, Ark, ED, Toone, EJ, and Zhou, P. "Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC." ACS Chem Biol 9.1 (January 17, 2014): 237-246.
PMID
24117400
Source
pubmed
Published In
ACS Chemical Biology
Volume
9
Issue
1
Publish Date
2014
Start Page
237
End Page
246
DOI
10.1021/cb400067g

Chasing acyl carrier protein through a catalytic cycle of lipid A production.

Acyl carrier protein represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon chains in vivo. Notably, type II acyl carrier proteins serve as a crucial interaction hub in primary cellular metabolism by communicating transiently between partner enzymes of the numerous biosynthetic pathways. However, the highly transient nature of such interactions and the inherent conformational mobility of acyl carrier protein have stymied previous attempts to visualize structurally acyl carrier protein tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but also of therapeutic value. For example, acyl carrier protein is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides in Gram-negative microorganisms, which is required for their growth and survival, and is an activator of the mammalian host's immune system, thus emerging as an important therapeutic target. During lipid A synthesis (Raetz pathway), acyl carrier protein shuttles acyl intermediates linked to its prosthetic 4'-phosphopantetheine group among four acyltransferases, including LpxD. Here we report the crystal structures of three forms of Escherichia coli acyl carrier protein engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures show the intricate interactions at the interface that optimally position acyl carrier protein for acyl delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled acyl carrier proteins provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4'-phosphopantetheine groups within the LpxD catalytic chamber shows an unprecedented role of acyl carrier protein in product release.

Authors
Masoudi, A; Raetz, CRH; Zhou, P; Pemble, CW
MLA Citation
Masoudi, A, Raetz, CRH, Zhou, P, and Pemble, CW. "Chasing acyl carrier protein through a catalytic cycle of lipid A production." Nature 505.7483 (January 16, 2014): 422-426.
PMID
24196711
Source
pubmed
Published In
Nature
Volume
505
Issue
7483
Publish Date
2014
Start Page
422
End Page
426
DOI
10.1038/nature12679

Chasing acyl carrier protein through a catalytic cycle of lipid A production

Acyl carrier protein represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon chains in vivo. Notably, type II acyl carrier proteins serve as a crucial interaction hub in primary cellular metabolism by communicating transiently between partner enzymes of the numerous biosynthetic pathways. However, the highly transient nature of such interactions and the inherent conformational mobility of acyl carrier protein have stymied previous attempts to visualize structurally acyl carrier protein tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but also of therapeutic value. For example, acyl carrier protein is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides in Gram-negative microorganisms, which is required for their growth and survival, and is an activator of the mammalian host's immune system, thus emerging as an important therapeutic target. During lipid A synthesis (Raetz pathway), acyl carrier protein shuttles acyl intermediates linked to its prosthetic 4′-phosphopantetheine group among four acyltransferases, including LpxD. Here we report the crystal structures of three forms of Escherichia coli acyl carrier protein engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures show the intricate interactions at the interface that optimally position acyl carrier protein for acyl delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled acyl carrier proteins provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber shows an unprecedented role of acyl carrier protein in product release. © 2014 Macmillan Publishers Limited. All rights reserved.

Authors
Masoudi, A; Raetz, CRH; Zhou, P; Pemble IV, CW
MLA Citation
Masoudi, A, Raetz, CRH, Zhou, P, and Pemble IV, CW. "Chasing acyl carrier protein through a catalytic cycle of lipid A production." Nature 505.7483 (January 1, 2014): 422-426.
Source
scopus
Published In
Nature
Volume
505
Issue
7483
Publish Date
2014
Start Page
422
End Page
426
DOI
10.1038/nature12679

Structural Basis of the Promiscuous Inhibitor Susceptibility of Escherichia coli LpxC

Authors
Lee, C-J; Liang, X; Gopalaswamy, R; Najeeb, J; Ark, ED; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Gopalaswamy, R, Najeeb, J, Ark, ED, Toone, EJ, and Zhou, P. "Structural Basis of the Promiscuous Inhibitor Susceptibility of Escherichia coli LpxC." ACS CHEMICAL BIOLOGY 9.1 (January 2014): 237-246.
Source
wos-lite
Published In
ACS Chemical Biology
Volume
9
Issue
1
Publish Date
2014
Start Page
237
End Page
246
DOI
10.1021/cb400067g

The UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis utilizes Mn2+ cluster for catalysis.

In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn(2+). EPR studies reveal the presence of a Mn(2+) cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn(2+) cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.

Authors
Young, HE; Donohue, MP; Smirnova, TI; Smirnov, AI; Zhou, P
MLA Citation
Young, HE, Donohue, MP, Smirnova, TI, Smirnov, AI, and Zhou, P. "The UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis utilizes Mn2+ cluster for catalysis." J Biol Chem 288.38 (September 20, 2013): 26987-27001.
PMID
23897835
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
38
Publish Date
2013
Start Page
26987
End Page
27001
DOI
10.1074/jbc.M113.497636

Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors.

The zinc-dependent deacetylase LpxC catalyzes the committed step of lipid A biosynthesis in Gram-negative bacteria and is a validated target for the development of novel antibiotics to combat multidrug-resistant Gram-negative infections. Many potent LpxC inhibitors contain an essential threonyl-hydroxamate headgroup for high-affinity interaction with LpxC. We report the synthesis, antibiotic activity, and structural and enzymatic characterization of novel LpxC inhibitors containing an additional aryl group in the threonyl-hydroxamate moiety, which expands the inhibitor-binding surface in LpxC. These compounds display enhanced potency against LpxC in enzymatic assays and superior antibiotic activity against Francisella novicida in cell culture. The comparison of the antibiotic activities of these compounds against a leaky Escherichia coli strain and the wild-type strain reveals the contribution of the formidable outer-membrane permeability barrier that reduces the compounds efficacy in cell culture and emphasizes the importance of maintaining a balanced hydrophobicity and hydrophilicity profile in developing effective LpxC-targeting antibiotics.

Authors
Liang, X; Lee, C-J; Zhao, J; Toone, EJ; Zhou, P
MLA Citation
Liang, X, Lee, C-J, Zhao, J, Toone, EJ, and Zhou, P. "Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors." J Med Chem 56.17 (September 12, 2013): 6954-6966.
PMID
23914798
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
56
Issue
17
Publish Date
2013
Start Page
6954
End Page
6966
DOI
10.1021/jm4007774

Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme.

Authors
Chung, BC; Zhao, J; Gillespie, RA; Kwon, D-Y; Guan, Z; Hong, J; Zhou, P; Lee, S-Y
MLA Citation
Chung, BC, Zhao, J, Gillespie, RA, Kwon, D-Y, Guan, Z, Hong, J, Zhou, P, and Lee, S-Y. "Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis." Science 341.6149 (August 30, 2013): 1012-1016.
PMID
23990562
Source
pubmed
Published In
Science
Volume
341
Issue
6149
Publish Date
2013
Start Page
1012
End Page
1016
DOI
10.1126/science.1236501

Storage of hydrogen spin polarization in long-lived 13C2 singlet order and implications for hyperpolarized magnetic resonance imaging.

Hyperpolarized magnetic resonance imaging (MRI) is a powerful technique enabling real-time monitoring of metabolites at concentration levels not accessible by standard MRI techniques. A considerable challenge this technique faces is the T1 decay of the hyperpolarization upon injection into the system under study. Here we show that A(n)A'(n)XX' spin systems such as (13)C2-1,2-diphenylacetylene ((13)C2-DPA) sustain long-lived polarization for both (13)C and (1)H spins with decay constants of almost 4.5 min at high magnetic fields of up to 16.44 T without spin-locking; the T1 of proton polarization is only 3.8 s. Therefore, storage of the proton polarization in a (13)C2-singlet state causes a 69-fold extension of the spin lifetime. Notably, this extension is demonstrated with proton-only pulse sequences, which can be readily implemented on standard clinical scanners.

Authors
Feng, Y; Theis, T; Liang, X; Wang, Q; Zhou, P; Warren, WS
MLA Citation
Feng, Y, Theis, T, Liang, X, Wang, Q, Zhou, P, and Warren, WS. "Storage of hydrogen spin polarization in long-lived 13C2 singlet order and implications for hyperpolarized magnetic resonance imaging." J Am Chem Soc 135.26 (July 3, 2013): 9632-9635.
PMID
23781874
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
135
Issue
26
Publish Date
2013
Start Page
9632
End Page
9635
DOI
10.1021/ja404936p

Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat.

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.

Authors
Liu, J; Fan, S; Lee, C-J; Greenleaf, AL; Zhou, P
MLA Citation
Liu, J, Fan, S, Lee, C-J, Greenleaf, AL, and Zhou, P. "Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat." J Biol Chem 288.15 (April 12, 2013): 10890-10901.
PMID
23436654
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
15
Publish Date
2013
Start Page
10890
End Page
10901
DOI
10.1074/jbc.M113.460238

Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis.

The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop-containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a precatalytic binding state, the ATP analogue AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg(2+)-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure.

Authors
Emptage, RP; Pemble, CW; York, JD; Raetz, CRH; Zhou, P
MLA Citation
Emptage, RP, Pemble, CW, York, JD, Raetz, CRH, and Zhou, P. "Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis." Biochemistry 52.13 (April 2, 2013): 2280-2290.
PMID
23464738
Source
pubmed
Published In
Biochemistry
Volume
52
Issue
13
Publish Date
2013
Start Page
2280
End Page
2290
DOI
10.1021/bi400097z

Specific interaction of the TCERG1 FF4-6 tandem repeat domains with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 of the CTD

Authors
Liu, J; Fan, S; Lee, C-J; Greenleaf, AL; Zhou, P
MLA Citation
Liu, J, Fan, S, Lee, C-J, Greenleaf, AL, and Zhou, P. "Specific interaction of the TCERG1 FF4-6 tandem repeat domains with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 of the CTD." April 2013.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
27
Publish Date
2013

Structural Basis for Acyl-substrate Delivery and Product Release by ACP in the Biosynthesis of Lipid A

Authors
Masoudi, A; Pemble, CW; Raetz, CRH; Zhou, P
MLA Citation
Masoudi, A, Pemble, CW, Raetz, CRH, and Zhou, P. "Structural Basis for Acyl-substrate Delivery and Product Release by ACP in the Biosynthesis of Lipid A." April 2013.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
27
Publish Date
2013

Mutants resistant to LpxC inhibitors by rebalancing cellular homeostasis.

LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in Escherichia coli, is an essential enzyme in virtually all gram-negative bacteria and is one of the most promising antibiotic targets for treatment of multidrug-resistant gram-negative infections. Despite the rapid development of LpxC-targeting antibiotics, the potential mechanisms of bacterial resistance to LpxC inhibitors remain poorly understood. Here, we report the isolation and biochemical characterization of spontaneously arising E. coli mutants that are over 200-fold more resistant to LpxC inhibitors than the wild-type strain. These mutants have two chromosomal point mutations that account for resistance additively and independently; one is in fabZ, a dehydratase in fatty acid biosynthesis; the other is in thrS, the Thr-tRNA ligase. For both enzymes, the isolated mutations result in reduced enzymatic activities in vitro. Unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring fabZ mutations in the absence of LpxC inhibitors, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain a balance between phospholipids and lipid A. Additionally, we show that the mutation in thrS slows protein production and cellular growth, indicating that reduced protein biosynthesis can confer a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal a previously unrecognized mechanism of antibiotic resistance by rebalancing cellular homeostasis.

Authors
Zeng, D; Zhao, J; Chung, HS; Guan, Z; Raetz, CRH; Zhou, P
MLA Citation
Zeng, D, Zhao, J, Chung, HS, Guan, Z, Raetz, CRH, and Zhou, P. "Mutants resistant to LpxC inhibitors by rebalancing cellular homeostasis." J Biol Chem 288.8 (February 22, 2013): 5475-5486.
PMID
23316051
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
8
Publish Date
2013
Start Page
5475
End Page
5486
DOI
10.1074/jbc.M112.447607

HASH: a program to accurately predict protein Hα shifts from neighboring backbone shifts.

Chemical shifts provide not only peak identities for analyzing nuclear magnetic resonance (NMR) data, but also an important source of conformational information for studying protein structures. Current structural studies requiring H(α) chemical shifts suffer from the following limitations. (1) For large proteins, the H(α) chemical shifts can be difficult to assign using conventional NMR triple-resonance experiments, mainly due to the fast transverse relaxation rate of C(α) that restricts the signal sensitivity. (2) Previous chemical shift prediction approaches either require homologous models with high sequence similarity or rely heavily on accurate backbone and side-chain structural coordinates. When neither sequence homologues nor structural coordinates are available, we must resort to other information to predict H(α) chemical shifts. Predicting accurate H(α) chemical shifts using other obtainable information, such as the chemical shifts of nearby backbone atoms (i.e., adjacent atoms in the sequence), can remedy the above dilemmas, and hence advance NMR-based structural studies of proteins. By specifically exploiting the dependencies on chemical shifts of nearby backbone atoms, we propose a novel machine learning algorithm, called HASH, to predict H(α) chemical shifts. HASH combines a new fragment-based chemical shift search approach with a non-parametric regression model, called the generalized additive model, to effectively solve the prediction problem. We demonstrate that the chemical shifts of nearby backbone atoms provide a reliable source of information for predicting accurate H(α) chemical shifts. Our testing results on different possible combinations of input data indicate that HASH has a wide rage of potential NMR applications in structural and biological studies of proteins.

Authors
Zeng, J; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Zhou, P, and Donald, BR. "HASH: a program to accurately predict protein Hα shifts from neighboring backbone shifts." J Biomol NMR 55.1 (January 2013): 105-118.
PMID
23242797
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
55
Issue
1
Publish Date
2013
Start Page
105
End Page
118
DOI
10.1007/s10858-012-9693-7

Extracting structural information from residual chemical shift anisotropy: Analytic solutions for peptide plane orientations and applications to determine protein structure

Residual dipolar coupling (RDC) and residual chemical shift anisotropy (RCSA) provide orientational restraints on internuclear vectors and the principal axes of chemical shift anisotropy (CSA) tensors, respectively. Mathematically, while an RDC represents a single sphero-conic, an RCSA can be interpreted as a linear combination of two sphero-conics. Since RDCs and RCSAs are described by a molecular alignment tensor, they contain inherent structural ambiguity due to the symmetry of the alignment tensor and the symmetry of the molecular fragment, which often leads to more than one orientation and conformation for the fragment consistent with the measured RDCs and RCSAs. While the orientational multiplicities have been long studied for RDCs, structural ambiguities arising from RCSAs have not been investigated. In this paper, we give exact and tight bounds on the number of peptide plane orientations consistent with multiple RDCs and/or RCSAs measured in one alignment medium. We prove that at most 16 orientations are possible for a peptide plane, which can be computed in closed form by solving a merely quadratic equation, and applying symmetry operations. Furthermore, we show that RCSAs can be used in the initial stages of structure determination to obtain highly accurate protein backbone global folds. We exploit the mathematical interplay between sphero-conics derived from RCSA and RDC, and protein kinematics, to derive quartic equations, which can be solved in closed-form to compute the protein backbone dihedral angles (φ, ψ). Building upon this, we designed a novel, sparse-data, polynomial-time divide-and-conquer algorithm to compute protein backbone conformations. Results on experimental NMR data for the protein human ubiquitin demonstrate that our algorithm computes backbone conformations with high accuracy from 13C′-RCSA or 15N-RCSA, and N-H N RDC data. We show that the structural information present in 13C′-RCSA and 15N-RCSA can be extracted analytically, and used in a rigorous algorithmic framework to compute a high-quality protein backbone global fold, from a limited amount of NMR data. This will benefit automated NOE assignment and high-resolution protein backbone structure determination from sparse NMR data. © 2013 Springer-Verlag.

Authors
Tripathy, C; Yan, AK; Zhou, P; Donald, BR
MLA Citation
Tripathy, C, Yan, AK, Zhou, P, and Donald, BR. "Extracting structural information from residual chemical shift anisotropy: Analytic solutions for peptide plane orientations and applications to determine protein structure." Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 7821 LNBI (2013): 271-284.
Source
scival
Published In
Lecture notes in computer science
Volume
7821 LNBI
Publish Date
2013
Start Page
271
End Page
284
DOI
10.1007/978-3-642-37195-0_25

Hash: A program to accurately predict protein Hα shifts from neighboring backbone shifts

Chemical shifts provide not only peak identities for analyzing nuclear magnetic resonance (NMR) data, but also an important source of conformational information for studying protein structures. Current structural studies requiring Hα chemical shifts suffer from the following limitations. (1) For large proteins, the Hα chemical shifts can be difficult to assign using conventional NMR triple-resonance experiments, mainly due to the fast transverse relaxation rate of Cα that restricts the signal sensitivity. (2) Previous chemical shift prediction approaches either require homologous models with high sequence similarity or rely heavily on accurate backbone and side-chain structural coordinates. When neither sequence homologues nor structural coordinates are available, we must resort to other information to predict Hα chemical shifts. Predicting accurate Hα chemical shifts using other obtainable information, such as the chemical shifts of nearby backbone atoms (i.e., adjacent atoms in the sequence), can remedy the above dilemmas, and hence advance NMR-based structural studies of proteins. By specifically exploiting the dependencies on chemical shifts of nearby backbone atoms, we propose a novel machine learning algorithm, called Hash, to predict Hα chemical shifts. Hash combines a new fragment-based chemical shift search approach with a non-parametric regression model, called the generalized additive model, to effectively solve the prediction problem. We demonstrate that the chemical shifts of nearby backbone atoms provide a reliable source of information for predicting accurate Hα chemical shifts. Our testing results on different possible combinations of input data indicate that Hash has a wide rage of potential NMR applications in structural and biological studies of proteins. © 2012 Springer Science+Business Media Dordrecht.

Authors
Zeng, J; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Zhou, P, and Donald, BR. "Hash: A program to accurately predict protein Hα shifts from neighboring backbone shifts." Journal of Biomolecular NMR 55.1 (2013): 105-118.
Source
scival
Published In
Journal of Biomolecular NMR
Volume
55
Issue
1
Publish Date
2013
Start Page
105
End Page
118
DOI
10.1007/s10858-012-9693-7

Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis

MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg2+ within the active site, and provide a structural basis of catalysis for this class of enzyme.

Authors
Chung, BC; Zhao, J; Gillespie, RA; Kwon, D-Y; Guan, Z; Hong, J; Zhou, P; Lee, S-Y
MLA Citation
Chung, BC, Zhao, J, Gillespie, RA, Kwon, D-Y, Guan, Z, Hong, J, Zhou, P, and Lee, S-Y. "Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis." Science 341.6149 (2013): 1012-1016.
Source
scival
Published In
Science
Volume
341
Issue
6149
Publish Date
2013
Start Page
1012
End Page
1016
DOI
10.1126/science.1240985

Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY.

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target's topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in (2)H/(13)C/(15)N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data.

Authors
Coggins, BE; Werner-Allen, JW; Yan, A; Zhou, P
MLA Citation
Coggins, BE, Werner-Allen, JW, Yan, A, and Zhou, P. "Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY." J Am Chem Soc 134.45 (November 14, 2012): 18619-18630.
PMID
22946863
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
134
Issue
45
Publish Date
2012
Start Page
18619
End Page
18630
DOI
10.1021/ja307445y

Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ.

DNA synthesis across lesions during genomic replication requires concerted actions of specialized DNA polymerases in a potentially mutagenic process known as translesion synthesis. Current models suggest that translesion synthesis in mammalian cells is achieved in two sequential steps, with a Y-family DNA polymerase (κ, η, ι, or Rev1) inserting a nucleotide opposite the lesion and with the heterodimeric B-family polymerase ζ, consisting of the catalytic Rev3 subunit and the accessory Rev7 subunit, replacing the insertion polymerase to carry out primer extension past the lesion. Effective translesion synthesis in vertebrates requires the scaffolding function of the C-terminal domain (CTD) of Rev1 that interacts with the Rev1-interacting region of polymerases κ, η, and ι and with the Rev7 subunit of polymerase ζ. We report the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ Rev1-interacting region. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a megatranslesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.

Authors
Wojtaszek, J; Lee, C-J; D'Souza, S; Minesinger, B; Kim, H; D'Andrea, AD; Walker, GC; Zhou, P
MLA Citation
Wojtaszek, J, Lee, C-J, D'Souza, S, Minesinger, B, Kim, H, D'Andrea, AD, Walker, GC, and Zhou, P. "Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ." J Biol Chem 287.40 (September 28, 2012): 33836-33846.
PMID
22859295
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
40
Publish Date
2012
Start Page
33836
End Page
33846
DOI
10.1074/jbc.M112.394841

Multifaceted recognition of vertebrate Rev1 by translesion polymerases ζ and κ.

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

Authors
Wojtaszek, J; Liu, J; D'Souza, S; Wang, S; Xue, Y; Walker, GC; Zhou, P
MLA Citation
Wojtaszek, J, Liu, J, D'Souza, S, Wang, S, Xue, Y, Walker, GC, and Zhou, P. "Multifaceted recognition of vertebrate Rev1 by translesion polymerases ζ and κ." J Biol Chem 287.31 (July 27, 2012): 26400-26408.
PMID
22700975
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
31
Publish Date
2012
Start Page
26400
End Page
26408
DOI
10.1074/jbc.M112.380998

1H, 13C and 15N backbone and side-chain resonance assignments of Drosophila melanogaster Ssu72

Ssu72 helps regulate transcription and co-transcriptional mRNA processing by dephosphorylating serine residues at the 5th position in the heptad repeats of the C-terminal domain of RNA polymerase II. Here we use multidimensional, multinuclear NMR experiments to assign the backbone and side-chain resonances of the 23 kDa Ssu72 from Drosophila melanogaster in the phosphate-bound state, and use NMR titrations to examine the phosphate-binding properties of three active site mutants. © 2011 Springer Science+Business Media B.V.

Authors
Werner-Allen, JW; Zhou, P
MLA Citation
Werner-Allen, JW, and Zhou, P. "1H, 13C and 15N backbone and side-chain resonance assignments of Drosophila melanogaster Ssu72." Biomolecular NMR Assignments 6.1 (April 1, 2012): 57-61.
Source
scopus
Published In
Biomolecular NMR Assignments
Volume
6
Issue
1
Publish Date
2012
Start Page
57
End Page
61
DOI
10.1007/s12104-011-9325-2

¹H, ¹³C and ¹⁵N backbone and side-chain resonance assignments of Drosophila melanogaster Ssu72.

Ssu72 helps regulate transcription and co-transcriptional mRNA processing by dephosphorylating serine residues at the 5th position in the heptad repeats of the C-terminal domain of RNA polymerase II. Here we use multidimensional, multinuclear NMR experiments to assign the backbone and side-chain resonances of the 23 kDa Ssu72 from Drosophila melanogaster in the phosphate-bound state, and use NMR titrations to examine the phosphate-binding properties of three active site mutants.

Authors
Werner-Allen, JW; Zhou, P
MLA Citation
Werner-Allen, JW, and Zhou, P. "¹H, ¹³C and ¹⁵N backbone and side-chain resonance assignments of Drosophila melanogaster Ssu72." Biomol NMR Assign 6.1 (April 2012): 57-61.
PMID
21732054
Source
pubmed
Published In
Biomolecular NMR Assignments
Volume
6
Issue
1
Publish Date
2012
Start Page
57
End Page
61
DOI
10.1007/s12104-011-9325-2

Structure-activity relationships of diacetylene-based LpxC inhibitors

Authors
Lee, C-J; Liang, X; Park, E; Zeng, D; Swanson, S; Chen, X; Nicholas, RA; Raetz, CRH; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Park, E, Zeng, D, Swanson, S, Chen, X, Nicholas, RA, Raetz, CRH, Toone, EJ, and Zhou, P. "Structure-activity relationships of diacetylene-based LpxC inhibitors." April 2012.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
26
Publish Date
2012

Enzymatic and structural studies of UDP-2,3-diacylglucosamine hydrolysis in lipid A biosynthesis

Authors
Young, HE; Metzger, LE; Lee, JK; Stroud, RM; Zhou, P; Raetz, CRH
MLA Citation
Young, HE, Metzger, LE, Lee, JK, Stroud, RM, Zhou, P, and Raetz, CRH. "Enzymatic and structural studies of UDP-2,3-diacylglucosamine hydrolysis in lipid A biosynthesis." April 2012.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
26
Publish Date
2012

Characterization of E. coli mutants resistant to LpxC inhibitors

Authors
Zeng, D; Zhou, P; Raetz, CRH
MLA Citation
Zeng, D, Zhou, P, and Raetz, CRH. "Characterization of E. coli mutants resistant to LpxC inhibitors." April 2012.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
26
Publish Date
2012

Protein loop closure using orientational restraints from NMR data.

Protein loops often play important roles in biological functions. Modeling loops accurately is crucial to determining the functional specificity of a protein. Despite the recent progress in loop prediction approaches, which led to a number of algorithms over the past decade, few rigorous algorithmic approaches exist to model protein loops using global orientational restraints, such as those obtained from residual dipolar coupling (RDC) data in solution nuclear magnetic resonance (NMR) spectroscopy. In this article, we present a novel, sparse data, RDC-based algorithm, which exploits the mathematical interplay between RDC-derived sphero-conics and protein kinematics, and formulates the loop structure determination problem as a system of low-degree polynomial equations that can be solved exactly, in closed-form. The polynomial roots, which encode the candidate conformations, are searched systematically, using provable pruning strategies that triage the vast majority of conformations, to enumerate or prune all possible loop conformations consistent with the data; therefore, completeness is ensured. Results on experimental RDC datasets for four proteins, including human ubiquitin, FF2, DinI, and GB3, demonstrate that our algorithm can compute loops with higher accuracy, a three- to six-fold improvement in backbone RMSD, versus those obtained by traditional structure determination protocols on the same data. Excellent results were also obtained on synthetic RDC datasets for protein loops of length 4, 8, and 12 used in previous studies. These results suggest that our algorithm can be successfully applied to determine protein loop conformations, and hence, will be useful in high-resolution protein backbone structure determination, including loops, from sparse NMR data. Proteins 2012. © 2011 Wiley Periodicals, Inc.

Authors
Tripathy, C; Zeng, J; Zhou, P; Donald, BR
MLA Citation
Tripathy, C, Zeng, J, Zhou, P, and Donald, BR. "Protein loop closure using orientational restraints from NMR data." Proteins 80.2 (February 2012): 433-453.
PMID
22161780
Source
pubmed
Published In
Proteins: Structure, Function and Bioinformatics
Volume
80
Issue
2
Publish Date
2012
Start Page
433
End Page
453
DOI
10.1002/prot.23207

Efficient acquisition of high-resolution 4-D diagonal-suppressed methyl-methyl NOESY for large proteins

The methyl-methyl NOESY experiment plays an important role in determining the global folds of large proteins. Despite the high sensitivity of this experiment, the analysis of methyl-methyl NOEs is frequently hindered by the limited chemical shift dispersion of methyl groups, particularly methyl protons. This makes it difficult to unambiguously assign all of the methyl-methyl NOE crosspeaks using 3-D spectroscopy. The recent development of sparse sampling methods enables highly efficient acquisition of high-resolution 4-D spectra, which provides an excellent solution to resolving the degeneracy of methyl signals. However, many reconstruction algorithms for processing sparsely-sampled NMR data do not provide adequate suppression of aliasing artifacts in the presence of strong NOE diagonal signals. In order to overcome this limitation, we present a 4-D diagonal-suppressed methyl-methyl NOESY experiment specifically optimized for ultrasparse sampling and evaluate it using a deuterated, ILV methyl-protonated sample of the 42 kDa Escherichia coli maltose binding protein (MBP). Suppression of diagonal signals removes the dynamic range barrier of the methyl-methyl NOESY experiment such that residual aliasing artifacts in the CLEAN-reconstructed high-resolution 4-D spectrum can be further reduced. At an ultrasparse sampling rate of less than 1%, we were able to identify and unambiguously assign the vast majority of expected NOE crosspeaks between methyl groups separated by less than 5 Å and to detect very weak NOE crosspeaks from methyl groups that are over 7 Å apart. © 2012 Elsevier Inc. All rights reserved.

Authors
Wen, J; Zhou, P; Wu, J
MLA Citation
Wen, J, Zhou, P, and Wu, J. "Efficient acquisition of high-resolution 4-D diagonal-suppressed methyl-methyl NOESY for large proteins." Journal of Magnetic Resonance 218 (2012): 128-132.
PMID
22464875
Source
scival
Published In
Journal of Magnetic Resonance
Volume
218
Publish Date
2012
Start Page
128
End Page
132
DOI
10.1016/j.jmr.2012.02.021

A Bayesian approach for determining protein side-chain rotamer conformations using unassigned NOE data.

A major bottleneck in protein structure determination via nuclear magnetic resonance (NMR) is the lengthy and laborious process of assigning resonances and nuclear Overhauser effect (NOE) cross peaks. Recent studies have shown that accurate backbone folds can be determined using sparse NMR data, such as residual dipolar couplings (RDCs) or backbone chemical shifts. This opens a question of whether we can also determine the accurate protein side-chain conformations using sparse or unassigned NMR data. We attack this question by using unassigned nuclear Overhauser effect spectroscopy (NOESY) data, which records the through-space dipolar interactions between protons nearby in three-dimensional (3D) space. We propose a Bayesian approach with a Markov random field (MRF) model to integrate the likelihood function derived from observed experimental data, with prior information (i.e., empirical molecular mechanics energies) about the protein structures. We unify the side-chain structure prediction problem with the side-chain structure determination problem using unassigned NMR data, and apply the deterministic dead-end elimination (DEE) and A* search algorithms to provably find the global optimum solution that maximizes the posterior probability. We employ a Hausdorff-based measure to derive the likelihood of a rotamer or a pairwise rotamer interaction from unassigned NOESY data. In addition, we apply a systematic and rigorous approach to estimate the experimental noise in NMR data, which also determines the weighting factor of the data term in the scoring function derived from the Bayesian framework. We tested our approach on real NMR data of three proteins: the FF Domain 2 of human transcription elongation factor CA150 (FF2), the B1 domain of Protein G (GB1), and human ubiquitin. The promising results indicate that our algorithm can be applied in high-resolution protein structure determination. Since our approach does not require any NOE assignment, it can accelerate the NMR structure determination process.

Authors
Zeng, J; Roberts, KE; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Roberts, KE, Zhou, P, and Donald, BR. "A Bayesian approach for determining protein side-chain rotamer conformations using unassigned NOE data." J Comput Biol 18.11 (November 2011): 1661-1679.
PMID
21970619
Source
pubmed
Published In
Journal of Computational Biology
Volume
18
Issue
11
Publish Date
2011
Start Page
1661
End Page
1679
DOI
10.1089/cmb.2011.0172

A geometric arrangement algorithm for structure determination of symmetric protein homo-oligomers from NOEs and RDCs.

Nuclear magnetic resonance (NMR) spectroscopy is a primary tool to perform structural studies of proteins in physiologically-relevant solution conditions. Restraints on distances between pairs of nuclei in the protein, derived from the nuclear Overhauser effect (NOE), provide information about the structure of the protein in its folded state. NMR studies of symmetric protein homo-oligomers present a unique challenge. Using X-filtered NOESY experiments, it is possible to determine whether an NOE restrains a pair of protons across different subunits or within a single subunit, but current experimental techniques are unable to determine in which subunits the restrained protons lie. Consequently, it is difficult to assign NOEs to particular pairs of subunits with certainty, thus hindering the structural analysis of the oligomeric state. Computational approaches are needed to address this subunit ambiguity, but traditional solutions often rely on stochastic search coupled with simulated annealing and simulations of simplified molecular dynamics, which have many tunable parameters that must be chosen carefully and can also fail to report structures consistent with the experimental restraints. In addition, these traditional approaches rarely provide guarantees on running time or solution quality. We reduce the structure determination of homo-oligomers with cyclic symmetry to computing geometric arrangements of unions of annuli in a plane. Our algorithm, disco, runs in expected O(n²) time, where n is the number of distance restraints, potentially assigned ambiguously. disco is guaranteed to report the exact set of oligomer structures consistent with the distance restraints and also with orientational restraints from residual dipolar couplings (RDCs). We demonstrate our method using two symmetric protein complexes: the trimeric E. coli diacylglycerol kinase (DAGK) and a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G (GB1). In both cases, disco computes oligomer structures with high precision and also finds distance restraints that are either mutually inconsistent or inconsistent with the RDCs. The entire protocol DISCO has been completely automated in a software package that is freely available and open-source at www.cs.duke.edu/donaldlab/software.php.

Authors
Martin, JW; Yan, AK; Bailey-Kellogg, C; Zhou, P; Donald, BR
MLA Citation
Martin, JW, Yan, AK, Bailey-Kellogg, C, Zhou, P, and Donald, BR. "A geometric arrangement algorithm for structure determination of symmetric protein homo-oligomers from NOEs and RDCs." J Comput Biol 18.11 (November 2011): 1507-1523.
PMID
22035328
Source
pubmed
Published In
Journal of Computational Biology
Volume
18
Issue
11
Publish Date
2011
Start Page
1507
End Page
1523
DOI
10.1089/cmb.2011.0173

Protein side-chain resonance assignment and NOE assignment using RDC-defined backbones without TOCSY data.

One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called NASCA: (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), NASCA: extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that NASCA: assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by NASCA: have backbone RMSD 0.8-1.5 Å from the reference structures determined by traditional NMR approaches.

Authors
Zeng, J; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Zhou, P, and Donald, BR. "Protein side-chain resonance assignment and NOE assignment using RDC-defined backbones without TOCSY data." J Biomol NMR 50.4 (August 2011): 371-395.
PMID
21706248
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
50
Issue
4
Publish Date
2011
Start Page
371
End Page
395
DOI
10.1007/s10858-011-9522-4

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of antichlamydial agents.

Authors
Nguyen, BD; Cunningham, D; Liang, X; Chen, X; Toone, EJ; Raetz, CRH; Zhou, P; Valdivia, RH
MLA Citation
Nguyen, BD, Cunningham, D, Liang, X, Chen, X, Toone, EJ, Raetz, CRH, Zhou, P, and Valdivia, RH. "Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis." Proc Natl Acad Sci U S A 108.25 (June 21, 2011): 10284-10289.
PMID
21628561
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
25
Publish Date
2011
Start Page
10284
End Page
10289
DOI
10.1073/pnas.1107478108

A graphical method for analyzing distance restraints using residual dipolar couplings for structure determination of symmetric protein homo-oligomers.

High-resolution structure determination of homo-oligomeric protein complexes remains a daunting task for NMR spectroscopists. Although isotope-filtered experiments allow separation of intermolecular NOEs from intramolecular NOEs and determination of the structure of each subunit within the oligomeric state, degenerate chemical shifts of equivalent nuclei from different subunits make it difficult to assign intermolecular NOEs to nuclei from specific pairs of subunits with certainty, hindering structural analysis of the oligomeric state. Here, we introduce a graphical method, DISCO, for the analysis of intermolecular distance restraints and structure determination of symmetric homo-oligomers using residual dipolar couplings. Based on knowledge that the symmetry axis of an oligomeric complex must be parallel to an eigenvector of the alignment tensor of residual dipolar couplings, we can represent distance restraints as annuli in a plane encoding the parameters of the symmetry axis. Oligomeric protein structures with the best restraint satisfaction correspond to regions of this plane with the greatest number of overlapping annuli. This graphical analysis yields a technique to characterize the complete set of oligomeric structures satisfying the distance restraints and to quantitatively evaluate the contribution of each distance restraint. We demonstrate our method for the trimeric E. coli diacylglycerol kinase, addressing the challenges in obtaining subunit assignments for distance restraints. We also demonstrate our method on a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G to show the resilience of our method to ambiguous atom assignments. In both studies, DISCO computed oligomer structures with high accuracy despite using ambiguously assigned distance restraints.

Authors
Martin, JW; Yan, AK; Bailey-Kellogg, C; Zhou, P; Donald, BR
MLA Citation
Martin, JW, Yan, AK, Bailey-Kellogg, C, Zhou, P, and Donald, BR. "A graphical method for analyzing distance restraints using residual dipolar couplings for structure determination of symmetric protein homo-oligomers." Protein Sci 20.6 (June 2011): 970-985.
PMID
21413097
Source
pubmed
Published In
Protein Science
Volume
20
Issue
6
Publish Date
2011
Start Page
970
End Page
985
DOI
10.1002/pro.620

cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72.

RNA polymerase II coordinates co-transcriptional events by recruiting distinct sets of nuclear factors to specific stages of transcription via changes of phosphorylation patterns along its C-terminal domain (CTD). Although it has become increasingly clear that proline isomerization also helps regulate CTD-associated processes, the molecular basis of its role is unknown. Here, we report the structure of the Ser(P)(5) CTD phosphatase Ssu72 in complex with substrate, revealing a remarkable CTD conformation with the Ser(P)(5)-Pro(6) motif in the cis configuration. We show that the cis-Ser(P)(5)-Pro(6) isomer is the minor population in solution and that Ess1-catalyzed cis-trans-proline isomerization facilitates rapid dephosphorylation by Ssu72, providing an explanation for recently discovered in vivo connections between these enzymes and a revised model for CTD-mediated small nuclear RNA termination. This work presents the first structural evidence of a cis-proline-specific enzyme and an unexpected mechanism of isomer-based regulation of phosphorylation, with broad implications for CTD biology.

Authors
Werner-Allen, JW; Lee, C-J; Liu, P; Nicely, NI; Wang, S; Greenleaf, AL; Zhou, P
MLA Citation
Werner-Allen, JW, Lee, C-J, Liu, P, Nicely, NI, Wang, S, Greenleaf, AL, and Zhou, P. "cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72." J Biol Chem 286.7 (February 18, 2011): 5717-5726.
PMID
21159777
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
7
Publish Date
2011
Start Page
5717
End Page
5726
DOI
10.1074/jbc.M110.197129

Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections.

Authors
Lee, C-J; Liang, X; Chen, X; Zeng, D; Joo, SH; Chung, HS; Barb, AW; Swanson, SM; Nicholas, RA; Li, Y; Toone, EJ; Raetz, CRH; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Chen, X, Zeng, D, Joo, SH, Chung, HS, Barb, AW, Swanson, SM, Nicholas, RA, Li, Y, Toone, EJ, Raetz, CRH, and Zhou, P. "Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design." Chem Biol 18.1 (January 28, 2011): 38-47.
PMID
21167751
Source
pubmed
Published In
Chemistry and Biology
Volume
18
Issue
1
Publish Date
2011
Start Page
38
End Page
47
DOI
10.1016/j.chembiol.2010.11.011

Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold.

Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.

Authors
Liang, X; Lee, C-J; Chen, X; Chung, HS; Zeng, D; Raetz, CRH; Li, Y; Zhou, P; Toone, EJ
MLA Citation
Liang, X, Lee, C-J, Chen, X, Chung, HS, Zeng, D, Raetz, CRH, Li, Y, Zhou, P, and Toone, EJ. "Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold." Bioorg Med Chem 19.2 (January 15, 2011): 852-860.
PMID
21194954
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
19
Issue
2
Publish Date
2011
Start Page
852
End Page
860
DOI
10.1016/j.bmc.2010.12.017

A Bayesian approach for determining protein side-chain rotamer conformations using unassigned NOE data

A major bottleneck in protein structure determination via nuclear magnetic resonance (NMR) is the lengthy and laborious process of assigning resonances and nuclear Overhauser effect (NOE) cross peaks. Recent studies have shown that accurate backbone folds can be determined using sparse NMR data, such as residual dipolar couplings (RDCs) or backbone chemical shifts. This opens a question of whether we can also determine the accurate protein side-chain conformations using sparse or unassigned NMR data. We attack this question by using unassigned nuclear Overhauser effect spectroscopy (NOESY) data, which record the through-space dipolar interactions between protons nearby in 3D space. We propose a Bayesian approach with a Markov random field (MRF) model to integrate the likelihood function derived from observed experimental data, with prior information (i.e., empirical molecular mechanics energies) about the protein structures. We unify the side-chain structure prediction problem with the side-chain structure determination problem using unassigned NMR data, and apply the deterministic dead-end elimination (DEE) and A*search algorithms to provably find the global optimum solution that maximizes the posterior probability. We employ a Hausdorff-based measure to derive the likelihood of a rotamer or a pairwise rotamer interaction from unassigned NOESY data. In addition, we apply a systematic and rigorous approach to estimate the experimental noise in NMR data, which also determines the weighting factor of the data term in the scoring function that is derived from the Bayesian framework. We tested our approach on real NMR data of three proteins, including the FF Domain 2 of human transcription elongation factor CA150 (FF2), the B1 domain of Protein G (GB1), and human ubiquitin. The promising results indicate that our approach can be applied in high-resolution protein structure determination. Since our approach does not require any NOE assignment, it can accelerate the NMR structure determination process. © 2011 Springer-Verlag.

Authors
Zeng, J; Roberts, KE; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Roberts, KE, Zhou, P, and Donald, BR. "A Bayesian approach for determining protein side-chain rotamer conformations using unassigned NOE data." Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 6577 LNBI (2011): 563-578.
Source
scival
Published In
Lecture notes in computer science
Volume
6577 LNBI
Publish Date
2011
Start Page
563
End Page
578
DOI
10.1007/978-3-642-20036-6_49

Protein loop closure using orientational restraints from NMR data

Protein loops often play important roles in biological functions such as binding, recognition, catalytic activities and allosteric regulation. Modeling loops that are biophysically sensible is crucial to determining the functional specificity of a protein. A variety of algorithms ranging from robotics-inspired inverse kinematics methods to fragmentbased homology modeling techniques have been developed to predict protein loops. However, determining the 3D structures of loops using global orientational restraints on internuclear vectors, such as those obtained from residual dipolar coupling (RDC) data in solution Nuclear Magnetic Resonance (NMR) spectroscopy, has not been well studied. In this paper, we present a novel algorithm that determines the protein loop conformations using a minimal amount of RDC data. Our algorithm exploits the interplay between the sphero-conics derived from RDCs and the protein kinematics, and formulates the loop structure determination problem as a system of low-degree polynomial equations that can be solved exactly and in closed form. The roots of these polynomial equations, which encode the candidate conformations, are searched systematically, using efficient and provable pruning strategies that triage the vast majority of conformations, to enumerate or prune all possible loop conformations consistent with the data. Our algorithm guarantees completeness by ensuring that a possible loop conformation consistent with the data is never missed. This data-driven algorithm provides a way to assess the structural quality from experimental data with minimal modeling assumptions. We applied our algorithm to compute the loops of human ubiquitin, the FF Domain 2 of human transcription elongation factor CA150 (FF2), the DNA damage inducible protein I (DinI) and the third IgG-binding domain of Protein G (GB3) from experimental RDC data. A comparison of our results versus those obtained by using traditional structure determination protocols on the same data shows that our algorithm is able to achieve higher accuracy: a 3- to 6-fold improvement in backbone RMSD. In addition, computational experiments on synthetic RDC data for a set of protein loops of length 4, 8 and 12 used in previous studies show that, whenever sparse RDCs can be measured, our algorithm can compute longer loops with high accuracy. These results demonstrate that our algorithm can be successfully applied to compute loops with high accuracy from a limited amount of NMR data. Our algorithm will be useful to determine high-quality complete protein backbone conformations, which will benefit the nuclear Overhauser effect (NOE) assignment process in high-resolution protein structure determination. © 2011 Springer-Verlag.

Authors
Tripathy, C; Zeng, J; Zhou, P; Donald, BR
MLA Citation
Tripathy, C, Zeng, J, Zhou, P, and Donald, BR. "Protein loop closure using orientational restraints from NMR data." Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 6577 LNBI (2011): 483-498.
Source
scival
Published In
Lecture notes in computer science
Volume
6577 LNBI
Publish Date
2011
Start Page
483
End Page
498
DOI
10.1007/978-3-642-20036-6_43

A geometric arrangement algorithm for structure determination of symmetric protein homo-oligomers from NOEs and RDCs

Nuclear magnetic resonance (NMR) spectroscopy is a primary tool to perform structural studies of proteins in the physiologically-relevant solution-state. Restraints on distances between pairs of nuclei in the protein, derived from the nuclear Overhauser effect (NOE) for example, provide information about the structure of the protein in its folded state. NMR studies of symmetric protein homo-oligomers present a unique challenge. Current techniques can determine whether an NOE restrains a pair of protons across different subunits or within a single subunit, but are unable to determine in which subunits the restrained protons lie. Consequently, it is difficult to assign NOEs to particular pairs of subunits with certainty, thus hindering the structural analysis of the oligomeric state. Hence, computational approaches are needed to address this subunit ambiguity. We reduce the structure determination of protein homo-oligomers with cyclic symmetry to computing geometric arrangements of unions of annuli in a plane. Our algorithm, disco, runs in expected O(n 2) time, where n is the number of distance restraints, and is guaranteed to report the exact set of oligomer structures consistent with ambiguously-assigned inter-subunit distance restraints and orientational restraints from residual dipolar couplings (RDCs). Since the symmetry axis of an oligomeric complex must be parallel to an eigenvector of the alignment tensor of RDCs, we can represent each distance restraint as a union of annuli in a plane encoding the configuration space of the symmetry axis. Oligomeric protein structures with the best restraint satisfaction correspond to faces of the arrangement contained in the greatest number of unions of annuli. We demonstrate our method using two symmetric protein complexes: the trimeric E. coli Diacylglycerol Kinase (DAGK), whose distance restraints possess at least two possible subunit assignments each; and a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G (GB1) using ambiguous NOEs. In both cases, disco computes oligomer structures with high accuracy. © 2011 Springer-Verlag.

Authors
Martin, JW; Yan, AK; Bailey-Kellogg, C; Zhou, P; Donald, BR
MLA Citation
Martin, JW, Yan, AK, Bailey-Kellogg, C, Zhou, P, and Donald, BR. "A geometric arrangement algorithm for structure determination of symmetric protein homo-oligomers from NOEs and RDCs." Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 6577 LNBI (2011): 222-237.
Source
scival
Published In
Lecture notes in computer science
Volume
6577 LNBI
Publish Date
2011
Start Page
222
End Page
237
DOI
10.1007/978-3-642-20036-6_21

ADAM9 inhibition increases membrane activity of ADAM10 and controls α-secretase processing of amyloid precursor protein

Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24-204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nM and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease.

Authors
Moss, ML; Powell, G; Miller, MA; Edwards, L; Qi, B; Sang, Q-XA; Strooper, BD; Tesseur, I; Lichtenthaler, SF; Taverna, M; Zhong, JL; Dingwall, C; Ferdous, T; Schlomann, U; Zhou, P; Griffith, LG; Lauffenburger, DA; Petrovich, R; Bartsch, JW
MLA Citation
Moss, ML, Powell, G, Miller, MA, Edwards, L, Qi, B, Sang, Q-XA, Strooper, BD, Tesseur, I, Lichtenthaler, SF, Taverna, M, Zhong, JL, Dingwall, C, Ferdous, T, Schlomann, U, Zhou, P, Griffith, LG, Lauffenburger, DA, Petrovich, R, and Bartsch, JW. "ADAM9 inhibition increases membrane activity of ADAM10 and controls α-secretase processing of amyloid precursor protein." Journal of Biological Chemistry 286.47 (2011): 40443-40451.
PMID
21956108
Source
scival
Published In
The Journal of biological chemistry
Volume
286
Issue
47
Publish Date
2011
Start Page
40443
End Page
40451
DOI
10.1074/jbc.M111.280495

Erratum to: NMR structure note: Solution structure of the core domain of MESD that is essential for proper folding of LRP5/6 (Journal of Biomolecular NMR (2010) 47 (283-288) DOI:10.1007/s10858-010-9426-8)

Authors
Chen, J; Li, Q; Liu, C-C; Zhou, P; Bu, G; Wang, J
MLA Citation
Chen, J, Li, Q, Liu, C-C, Zhou, P, Bu, G, and Wang, J. "Erratum to: NMR structure note: Solution structure of the core domain of MESD that is essential for proper folding of LRP5/6 (Journal of Biomolecular NMR (2010) 47 (283-288) DOI:10.1007/s10858-010-9426-8)." Journal of Biomolecular NMR 50.1 (2011): 101--.
Source
scival
Published In
Journal of Biomolecular NMR
Volume
50
Issue
1
Publish Date
2011
Start Page
101-
DOI
10.1007/s10858-011-9476-6

Sparsely sampled high-resolution 4-D experiments for efficient backbone resonance assignment of disordered proteins

Intrinsically disordered proteins (IDPs) play important roles in many critical cellular processes. Due to their limited chemical shift dispersion, IDPs often require four pairs of resonance connectivities (Hα, Cα, Cβ and CO) for establishing sequential backbone assignment. Because most conventional 4-D triple-resonance experiments share an overlapping Cα evolution period, combining existing 4-D experiments does not offer an optimal solution for non-redundant collection of a complete set of backbone resonances. Using alternative chemical shift evolution schemes, we propose a new pair of 4-D triple-resonance experiments - HA(CA)CO(CA)NH/HA(CA)CONH - that complement the 4-D HNCACB/HN(CO)CACB experiments to provide complete backbone resonance information. Collection of high-resolution 4-D spectra with sparse sampling and FFT-CLEAN processing enables efficient acquisition and assignment of complete backbone resonances of IDPs. Importantly, because the CLEAN procedure iteratively identifies resonance signals and removes their associating aliasing artifacts, it greatly reduces the dependence of the reconstruction quality on sampling schemes and produces high-quality spectra even with less-than-optimal sampling schemes. © 2010 Elsevier Inc. All rights reserved.

Authors
Wen, J; Wu, J; Zhou, P
MLA Citation
Wen, J, Wu, J, and Zhou, P. "Sparsely sampled high-resolution 4-D experiments for efficient backbone resonance assignment of disordered proteins." Journal of Magnetic Resonance 209.1 (2011): 94-100.
PMID
21277815
Source
scival
Published In
Journal of Magnetic Resonance
Volume
209
Issue
1
Publish Date
2011
Start Page
94
End Page
100
DOI
10.1016/j.jmr.2010.12.012

Radial sampling for fast NMR: Concepts and practices over three decades.

Authors
Coggins, BE; Venters, RA; Zhou, P
MLA Citation
Coggins, BE, Venters, RA, and Zhou, P. "Radial sampling for fast NMR: Concepts and practices over three decades." Prog Nucl Magn Reson Spectrosc 57.4 (November 2010): 381-419. (Review)
PMID
20920757
Source
pubmed
Published In
Progress in Nuclear Magnetic Resonance Spectroscopy
Volume
57
Issue
4
Publish Date
2010
Start Page
381
End Page
419
DOI
10.1016/j.pnmrs.2010.07.001

NMR structure note: solution structure of the core domain of MESD that is essential for proper folding of LRP5/6.

Authors
Chen, J; Li, Q; Liu, C-C; Zhou, P; Bu, G; Wang, J
MLA Citation
Chen, J, Li, Q, Liu, C-C, Zhou, P, Bu, G, and Wang, J. "NMR structure note: solution structure of the core domain of MESD that is essential for proper folding of LRP5/6." Journal of biomolecular NMR 47.4 (August 2010): 283-288.
PMID
20506034
Source
epmc
Published In
Journal of Biomolecular NMR
Volume
47
Issue
4
Publish Date
2010
Start Page
283
End Page
288
DOI
10.1007/s10858-010-9426-8

Fast acquisition of high resolution 4-D amide-amide NOESY with diagonal suppression, sparse sampling and FFT-CLEAN.

Amide-amide NOESY provides important distance constraints for calculating global folds of large proteins, especially integral membrane proteins with beta-barrel folds. Here, we describe a diagonal-suppressed 4-D NH-NH TROSY-NOESY-TROSY (ds-TNT) experiment for NMR studies of large proteins. The ds-TNT experiment employs a spin state selective transfer scheme that suppresses diagonal signals while providing TROSY optimization in all four dimensions. Active suppression of the strong diagonal peaks greatly reduces the dynamic range of observable signals, making this experiment particularly suitable for use with sparse sampling techniques. To demonstrate the utility of this method, we collected a high resolution 4-D ds-TNT spectrum of a 23kDa protein using randomized concentric shell sampling (RCSS), and we used FFT-CLEAN processing for further reduction of aliasing artifacts - the first application of these techniques to a NOESY experiment. A comparison of peak parameters in the high resolution 4-D dataset with those from a conventionally-sampled 3-D control spectrum shows an accurate reproduction of NOE crosspeaks in addition to a significant reduction in resonance overlap, which largely eliminates assignment ambiguity. Likewise, a comparison of 4-D peak intensities and volumes before and after application of the CLEAN procedure demonstrates that the reduction of aliasing artifacts by CLEAN does not systematically distort NMR signals.

Authors
Werner-Allen, JW; Coggins, BE; Zhou, P
MLA Citation
Werner-Allen, JW, Coggins, BE, and Zhou, P. "Fast acquisition of high resolution 4-D amide-amide NOESY with diagonal suppression, sparse sampling and FFT-CLEAN." J Magn Reson 204.1 (May 2010): 173-178.
PMID
20227311
Source
pubmed
Published In
Journal of Magnetic Resonance
Volume
204
Issue
1
Publish Date
2010
Start Page
173
End Page
178
DOI
10.1016/j.jmr.2010.02.017

Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240.

The UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A biosynthesis, an essential pathway in Gram-negative bacteria. We report the backbone resonance assignments of the 34 kDa LpxC from Escherichia coli in complex with the antibiotic L-161,240 using multidimensional, multinuclear NMR experiments. The (1)H chemical shifts of complexed L-161,240 are also determined.

Authors
Barb, AW; Jiang, L; Raetz, CRH; Zhou, P
MLA Citation
Barb, AW, Jiang, L, Raetz, CRH, and Zhou, P. "Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240." Biomol NMR Assign 4.1 (April 2010): 37-40.
PMID
19941092
Source
pubmed
Published In
Biomolecular NMR Assignments
Volume
4
Issue
1
Publish Date
2010
Start Page
37
End Page
40
DOI
10.1007/s12104-009-9201-5

Structure-activity relationships of diacetylene-based LpxC inhibitors

Authors
Lee, C-J; Liang, X; Park, E; Zeng, D; Swanson, S; Chen, X; Nicholas, RA; Raetz, CRH; Toone, EJ; Zhou, P
MLA Citation
Lee, C-J, Liang, X, Park, E, Zeng, D, Swanson, S, Chen, X, Nicholas, RA, Raetz, CRH, Toone, EJ, and Zhou, P. "Structure-activity relationships of diacetylene-based LpxC inhibitors." FASEB JOURNAL 24 (April 2010).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
24
Publish Date
2010

Enzymatic and structural studies of UDP-2,3-diacylglucosamine hydrolysis in lipid A biosynthesis

Authors
Young, HE; Metzger, LE; Lee, JK; Stroud, RM; Zhou, P; Raetz, CRH
MLA Citation
Young, HE, Metzger, LE, Lee, JK, Stroud, RM, Zhou, P, and Raetz, CRH. "Enzymatic and structural studies of UDP-2,3-diacylglucosamine hydrolysis in lipid A biosynthesis." FASEB JOURNAL 24 (April 2010).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
24
Publish Date
2010

Characterization of E. coli mutants resistant to LpxC inhibitors

Authors
Zeng, D; Zhou, P; Raetz, CRH
MLA Citation
Zeng, D, Zhou, P, and Raetz, CRH. "Characterization of E. coli mutants resistant to LpxC inhibitors." FASEB JOURNAL 24 (April 2010).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
24
Publish Date
2010

Unconventional ubiquitin recognition by the ubiquitin-binding motif within the Y family DNA polymerases iota and Rev1.

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) iota and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol iota and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A, of ubiquitin abolishes UBM binding. Human pol iota contains two functional UBMs, both contributing to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol iota in replication foci and Rev1-mediated DNA damage tolerance in vivo.

Authors
Bomar, MG; D'Souza, S; Bienko, M; Dikic, I; Walker, GC; Zhou, P
MLA Citation
Bomar, MG, D'Souza, S, Bienko, M, Dikic, I, Walker, GC, and Zhou, P. "Unconventional ubiquitin recognition by the ubiquitin-binding motif within the Y family DNA polymerases iota and Rev1." Mol Cell 37.3 (February 12, 2010): 408-417.
PMID
20159559
Source
pubmed
Published In
Molecular Cell
Volume
37
Issue
3
Publish Date
2010
Start Page
408
End Page
417
DOI
10.1016/j.molcel.2009.12.038

Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies.

Although the rapid progress of NMR technology has significantly expanded the range of NMR-trackable systems, preparation of NMR-suitable samples that are highly soluble and stable remains a bottleneck for studies of many biological systems. The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed unapproachable by solution NMR techniques. In this review, we provide a brief survey of the development and successful applications of the SET strategy in biomolecular NMR.We also comment on the criteria for choosing optimal SETs, such as for differently charged target proteins, and recent new developments on NMR-invisible SETs.

Authors
Zhou, P; Wagner, G
MLA Citation
Zhou, P, and Wagner, G. "Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies." J Biomol NMR 46.1 (January 2010): 23-31.
PMID
19731047
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
46
Issue
1
Publish Date
2010
Start Page
23
End Page
31
DOI
10.1007/s10858-009-9371-6

Algorithms and analytic solutions using sparse residual dipolar couplings for high-resolution automated protein backbone structure determination by NMR

Developing robust and automated protein structure determination algorithms using nuclear magnetic resonance (NMR) data is an important goal in computational structural biology. Algorithms based on global orientational restraints from residual dipolar couplings (RDCs) promise to be quicker and more accurate than approaches that use only distance restraints. Recent development of analytic expressions for the roots of RDC equations together with protein kinematics has enabled exact, linear-time algorithms, highly desirable over earlier stochastic methods. In addition to providing guarantees on the number and quality of solutions, exact algorithms require a minimal amount of NMR data, thereby reducing the number of NMR experiments. Implementations of these methods determine the solution structures by explicitly computing the intersections of algebraic curves representing discrete RDC values. However, if additional RDC data can be measured, the algebraic curves no longer generically intersect. We address this situation in the paper and show that globally optimal structures can still be computed analytically as points closest to all of the algebraic curves representing the RDCs. We present new algorithms that expand the types and number of RDCs from which analytic solutions are computed. We evaluate the performance of our algorithms on NMR data for four proteins: human ubiquitin, DNA-damage-inducible protein I (DinI), the Z domain of staphylococcal protein A (SpA), and the third IgG-binding domain of Protein G (GB3). The results show that our algorithms are able to determine high-resolution backbone structures from a limited amount of NMR data. © 2010 Springer-Verlag Berlin Heidelberg.

Authors
Yershova, A; Tripathy, C; Zhou, P; Donald, BR
MLA Citation
Yershova, A, Tripathy, C, Zhou, P, and Donald, BR. "Algorithms and analytic solutions using sparse residual dipolar couplings for high-resolution automated protein backbone structure determination by NMR." Springer Tracts in Advanced Robotics 68.STAR (2010): 355-372.
Source
scival
Published In
Springer Tracts in Advanced Robotics
Volume
68
Issue
STAR
Publish Date
2010
Start Page
355
End Page
372
DOI
10.1007/978-3-642-17452-0_21

A Markov random field framework for protein side-chain resonance assignment

Nuclear magnetic resonance (NMR) spectroscopy plays a critical role in structural genomics, and serves as a primary tool for determining protein structures, dynamics and interactions in physiologically-relevant solution conditions. The current speed of protein structure determination via NMR is limited by the lengthy time required in resonance assignment, which maps spectral peaks to specific atoms and residues in the primary sequence. Although numerous algorithms have been developed to address the backbone resonance assignment problem [68,2,10,37,14,64,1,31,60], little work has been done to automate side-chain resonance assignment [43, 48, 5]. Most previous attempts in assigning side-chain resonances depend on a set of NMR experiments that record through-bond interactions with side-chain protons for each residue. Unfortunately, these NMR experiments have low sensitivity and limited performance on large proteins, which makes it difficult to obtain enough side-chain resonance assignments. On the other hand, it is essential to obtain almost all of the side-chain resonance assignments as a prerequisite for high-resolution structure determination. To overcome this deficiency, we present a novel side-chain resonance assignment algorithm based on alternative NMR experiments measuring through-space interactions between protons in the protein, which also provide crucial distance restraints and are normally required in high-resolution structure determination. We cast the sidechain resonance assignment problem into a Markov Random Field (MRF) framework, and extend and apply combinatorial protein design algorithms to compute the optimal solution that best interprets the NMR data. Our MRF framework captures the contact map information of the protein derived from NMR spectra, and exploits the structural information available from the backbone conformations determined by orientational restraints and a set of discretized side-chain conformations (i.e., rotamers). A Hausdorff-based computation is employed in the scoring function to evaluate the probability of side-chain resonance assignments to generate the observed NMR spectra. The complexity of the assignment problem is first reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is used to find a set of optimal side-chain resonance assignments that best fit the NMR data. We have tested our algorithm on NMR data for five proteins, including the FF Domain 2 of human transcription elongation factor CA150 (FF2), the B1 domain of Protein G (GB1), human ubiquitin, the ubiquitin-binding zinc finger domain of the human Y-family DNA polymerase Eta (pol η UBZ), and the human Set2-Rpb1 interacting domain (hSRI). Our algorithm assigns resonances for more than 90% of the protons in the proteins, and achieves about 80% correct side-chain resonance assignments. The final structures computed using distance restraints resulting from the set of assigned side-chain resonances have backbone RMSD 0.5-1.4 Å and all-heavyatom RMSD 1.0 - 2.2 Å from the reference structures that were determined by X-ray crystallography or traditional NMR approaches. These results demonstrate that our algorithm can be successfully applied to automate side-chain resonance assignment and high-quality protein structure determination. Since our algorithm does not require any specific NMR experiments for measuring the through-bond interactions with side-chain protons, it can save a significant amount of both experimental cost and spectrometer time, and hence accelerate the NMR structure determination process. © Springer-Verlag Berlin Heidelberg 2010.

Authors
Zeng, J; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Zhou, P, and Donald, BR. "A Markov random field framework for protein side-chain resonance assignment." Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 6044 LNBI (2010): 550-570.
Source
scival
Published In
Lecture notes in computer science
Volume
6044 LNBI
Publish Date
2010
Start Page
550
End Page
570
DOI
10.1007/978-3-642-12683-3_36

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar KD values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins. © 2010 American Chemical Society.

Authors
Hard, RL; Liu, J; Shen, J; Zhou, P; Pei, D
MLA Citation
Hard, RL, Liu, J, Shen, J, Zhou, P, and Pei, D. "HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins." Biochemistry 49.50 (2010): 10737-10746.
Website
http://hdl.handle.net/10161/4013
PMID
21090589
Source
scival
Published In
Biochemistry
Volume
49
Issue
50
Publish Date
2010
Start Page
10737
End Page
10746
DOI
10.1021/bi101014s

The unusual UBZ domain of Saccharomyces cerevisiae polymerase η

Recent research has revealed the presence of ubiquitin-binding domains in the Y family polymerases. The ubiquitin-binding zinc finger (UBZ) domain of human polymerase η is vital for its regulation, localization, and function. Here, we elucidate structural and functional features of the non-canonical UBZ motif of Saccharomyces cerevisiae pol η. Characterization of pol η mutants confirms the importance of the UBZ motif and implies that its function is independent of zinc binding. Intriguingly, we demonstrate that zinc does bind to and affect the structure of the purified UBZ domain, but is not required for its ubiquitin-binding activity. Our finding that this unusual zinc finger is able to interact with ubiquitin even in its apo form adds support to the model that ubiquitin binding is the primary and functionally important activity of the UBZ domain in S. cerevisiae polymerase η. Putative ubiquitin-binding domains, primarily UBZs, are identified in the majority of known pol η homologs. We discuss the implications of our observations for zinc finger structure and pol η regulation. © 2010 Elsevier B.V.

Authors
Woodruff, RV; Bomar, MG; D'Souza, S; Zhou, P; Walker, GC
MLA Citation
Woodruff, RV, Bomar, MG, D'Souza, S, Zhou, P, and Walker, GC. "The unusual UBZ domain of Saccharomyces cerevisiae polymerase η." DNA Repair 9.11 (2010): 1130-1141.
PMID
20837403
Source
scival
Published In
DNA Repair
Volume
9
Issue
11
Publish Date
2010
Start Page
1130
End Page
1141
DOI
10.1016/j.dnarep.2010.08.001

High-resolution protein structure determination starting with a global fold calculated from exact solutions to the RDC equations.

We present a novel structure determination approach that exploits the global orientational restraints from RDCs to resolve ambiguous NOE assignments. Unlike traditional approaches that bootstrap the initial fold from ambiguous NOE assignments, we start by using RDCs to compute accurate secondary structure element (SSE) backbones at the beginning of structure calculation. Our structure determination package, called RDC-PANDA: (RDC-based SSE PAcking with NOEs for Structure Determination and NOE Assignment), consists of three modules: (1) RDC-EXACT: ; (2) PACKER: ; and (3) HANA: (HAusdorff-based NOE Assignment). RDC-EXACT: computes the global optimal solution of backbone dihedral angles for each secondary structure element by exactly solving a system of quartic RDC equations derived by Wang and Donald (Proceedings of the IEEE computational systems bioinformatics conference (CSB), Stanford, CA, 2004a; J Biomol NMR 29(3):223-242, 2004b), and systematically searching over the roots, each of which is a backbone dihedral varphi- or psi-angle consistent with the RDC data. Using a small number of unambiguous inter-SSE NOEs extracted using only chemical shift information, PACKER: performs a systematic search for the core structure, including all SSE backbone conformations. HANA: uses a Hausdorff-based scoring function to measure the similarity between the experimental spectra and the back-computed NOE pattern for each side-chain from a statistically-diverse rotamer library, and drives the selection of optimal position-specific rotamers for filtering ambiguous NOE assignments. Finally, a local minimization approach is used to compute the loops and refine side-chain conformations by fixing the core structure as a rigid body while allowing movement of loops and side-chains. RDC-PANDA: was applied to NMR data for the FF Domain 2 of human transcription elongation factor CA150 (RNA polymerase II C-terminal domain interacting protein), human ubiquitin, the ubiquitin-binding zinc finger domain of the human Y-family DNA polymerase Eta (pol eta UBZ), and the human Set2-Rpb1 interacting domain (hSRI). These results demonstrated the efficiency and accuracy of our algorithm, and show that RDC-PANDA: can be successfully applied for high-resolution protein structure determination using only a limited set of NMR data by first computing RDC-defined backbones.

Authors
Zeng, J; Boyles, J; Tripathy, C; Wang, L; Yan, A; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Boyles, J, Tripathy, C, Wang, L, Yan, A, Zhou, P, and Donald, BR. "High-resolution protein structure determination starting with a global fold calculated from exact solutions to the RDC equations." J Biomol NMR 45.3 (November 2009): 265-281.
PMID
19711185
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
45
Issue
3
Publish Date
2009
Start Page
265
End Page
281
DOI
10.1007/s10858-009-9366-3

Uridine-based inhibitors as new leads for antibiotics targeting Escherichia coli LpxC.

The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A (endotoxin) biosynthesis in Gram-negative bacteria and is a validated antibiotic target. Although several previously described compounds bind to the unique acyl chain binding passage of LpxC with high affinity, strategies to target the enzyme's UDP-binding site have not been reported. Here the identification of a series of uridine-based LpxC inhibitors is presented. The most potent examined, 1-68A, is a pH-dependent, two-step, covalent inhibitor of Escherichia coli LpxC that competes with UDP to bind the enzyme in the first step of inhibition. Compound 1-68A exhibits a K(I) of 54 muM and a maximal rate of inactivation (k(inact)) of 1.7 min(-1) at pH 7.4. Dithiothreitol, glutathione and the C207A mutant of E. coli LpxC prevent the formation of a covalent complex by 1-68A, suggesting a role for Cys-207 in inhibition. The inhibitory activity of 1-68A and a panel of synthetic analogues identified moieties necessary for inhibition. 1-68A and a 2-dehydroxy analogue, 1-68Aa, inhibit several purified LpxC orthologues. These compounds may provide new scaffolds for extension of existing LpxC-inhibiting antibiotics to target the UDP binding pocket.

Authors
Barb, AW; Leavy, TM; Robins, LI; Guan, Z; Six, DA; Zhou, P; Hangauer, MJ; Bertozzi, CR; Raetz, CRH
MLA Citation
Barb, AW, Leavy, TM, Robins, LI, Guan, Z, Six, DA, Zhou, P, Hangauer, MJ, Bertozzi, CR, and Raetz, CRH. "Uridine-based inhibitors as new leads for antibiotics targeting Escherichia coli LpxC." Biochemistry 48.14 (April 14, 2009): 3068-3077.
PMID
19256534
Source
pubmed
Published In
Biochemistry
Volume
48
Issue
14
Publish Date
2009
Start Page
3068
End Page
3077
DOI
10.1021/bi900167q

Correction to uridine-based inhibitors as new leads for antibiotics targeting Escherichia coli LpxC (Biochemistry (2009) 48, (3068) DOI: 10.1021/bi900167q)

Authors
Barb, AW; Leavy, TM; Robins, LI; Guan, Z; Six, DA; Zhou, P; Hangauer, MJ; Bertozzi, CR; Raetz, C
MLA Citation
Barb, AW, Leavy, TM, Robins, LI, Guan, Z, Six, DA, Zhou, P, Hangauer, MJ, Bertozzi, CR, and Raetz, C. "Correction to uridine-based inhibitors as new leads for antibiotics targeting Escherichia coli LpxC (Biochemistry (2009) 48, (3068) DOI: 10.1021/bi900167q)." Biochemistry 48.32 (2009): 7776--.
Source
scival
Published In
Biochemistry
Volume
48
Issue
32
Publish Date
2009
Start Page
7776-
DOI
10.1021/bi9012066

High resolution 4-D spectroscopy with sparse concentric shell sampling and FFT-CLEAN.

Recent efforts to reduce the measurement time for multidimensional NMR experiments have fostered the development of a variety of new procedures for sampling and data processing. We recently described concentric ring sampling for 3-D NMR experiments, which is superior to radial sampling as input for processing by a multidimensional discrete Fourier transform. Here, we report the extension of this approach to 4-D spectroscopy as Randomized Concentric Shell Sampling (RCSS), where sampling points for the indirect dimensions are positioned on concentric shells, and where random rotations in the angular space are used to avoid coherent artifacts. With simulations, we show that RCSS produces a very low level of artifacts, even with a very limited number of sampling points. The RCSS sampling patterns can be adapted to fine rectangular grids to permit use of the Fast Fourier Transform in data processing, without an apparent increase in the artifact level. These artifacts can be further reduced to the noise level using the iterative CLEAN algorithm developed in radioastronomy. We demonstrate these methods on the high resolution 4-D HCCH-TOCSY spectrum of protein G's B1 domain, using only 1.2% of the sampling that would be needed conventionally for this resolution. The use of a multidimensional FFT instead of the slow DFT for initial data processing and for subsequent CLEAN significantly reduces the calculation time, yielding an artifact level that is on par with the level of the true spectral noise.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "High resolution 4-D spectroscopy with sparse concentric shell sampling and FFT-CLEAN." J Biomol NMR 42.4 (December 2008): 225-239.
PMID
18853260
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
42
Issue
4
Publish Date
2008
Start Page
225
End Page
239
DOI
10.1007/s10858-008-9275-x

Mechanism and inhibition of LpxC: an essential zinc-dependent deacetylase of bacterial lipid A synthesis.

Multi-drug resistant (MDR), pathogenic Gram-negative bacteria pose a serious health threat, and novel antibiotic targets must be identified to combat MDR infections. One promising target is the zinc-dependent metalloamidase UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyzes the committed step of lipid A (endotoxin) biosynthesis. LpxC is an essential, single copy gene that is conserved in virtually all Gram-negative bacteria. LpxC structures, revealed by NMR and X-ray crystallography, demonstrate that LpxC adopts a novel 'beta-alpha-alpha-beta sandwich' fold and encapsulates the acyl chain of the substrate with a unique hydrophobic passage. Kinetic analysis revealed that LpxC functions by a general acid-base mechanism, with a glutamate serving as the general base. Many potent LpxC inhibitors have been identified, and most contain a hydroxamate group targeting the catalytic zinc ion. Although early LpxC-inhibitors were either narrow-spectrum antibiotics or broad-spectrum in vitro LpxC inhibitors with limited antibiotic properties, the recently discovered compound CHIR-090 is a powerful antibiotic that controls the growth of Escherichia coli and Pseudomonas aeruginosa, with an efficacy rivaling that of the FDA-approved antibiotic ciprofloxacin. CHIR-090 inhibits a wide range of LpxC enzymes with sub-nanomolar affinity in vitro, and is a two-step, slow, tight-binding inhibitor of Aquifex aeolicus and E. coli LpxC. The success of CHIR-090 suggests that potent LpxC-targeting antibiotics may be developed to control a broad range of Gram-negative bacteria.

Authors
Barb, AW; Zhou, P
MLA Citation
Barb, AW, and Zhou, P. "Mechanism and inhibition of LpxC: an essential zinc-dependent deacetylase of bacterial lipid A synthesis." Curr Pharm Biotechnol 9.1 (February 2008): 9-15. (Review)
PMID
18289052
Source
pubmed
Published In
Current Pharmaceutical Biotechnology
Volume
9
Issue
1
Publish Date
2008
Start Page
9
End Page
15

A HAUSDORFF-BASED NOE ASSIGNMENT ALGORITHM USING PROTEIN BACKBONE DETERMINED FROM RESIDUAL DIPOLAR COUPLINGS AND ROTAMER PATTERNS.

High-throughput structure determination based on solution Nuclear Magnetic Resonance (NMR) spectroscopy plays an important role in structural genomics. One of the main bottlenecks in NMR structure determination is the interpretation of NMR data to obtain a sufficient number of accurate distance restraints by assigning nuclear Overhauser effect (NOE) spectral peaks to pairs of protons. The difficulty in automated NOE assignment mainly lies in the ambiguities arising both from the resonance degeneracy of chemical shifts and from the uncertainty due to experimental errors in NOE peak positions. In this paper we present a novel NOE assignment algorithm, called HAusdorff-based NOE Assignment (HANA), that starts with a high-resolution protein backbone computed using only two residual dipolar couplings (RDCs) per residue37, 39, employs a Hausdorff-based pattern matching technique to deduce similarity between experimental and back-computed NOE spectra for each rotamer from a statistically diverse library, and drives the selection of optimal position-specific rotamers for filtering ambiguous NOE assignments. Our algorithm runs in time O(tn(3) +tn log t), where t is the maximum number of rotamers per residue and n is the size of the protein. Application of our algorithm on biological NMR data for three proteins, namely, human ubiquitin, the zinc finger domain of the human DNA Y-polymerase Eta (pol η) and the human Set2-Rpb1 interacting domain (hSRI) demonstrates that our algorithm overcomes spectral noise to achieve more than 90% assignment accuracy. Additionally, the final structures calculated using our automated NOE assignments have backbone RMSD < 1.7 Å and all-heavy-atom RMSD < 2.5 Å from reference structures that were determined either by X-ray crystallography or traditional NMR approaches. These results show that our NOE assignment algorithm can be successfully applied to protein NMR spectra to obtain high-quality structures.

Authors
Zeng, JM; Tripathy, C; Zhou, P; Donald, BR
MLA Citation
Zeng, JM, Tripathy, C, Zhou, P, and Donald, BR. "A HAUSDORFF-BASED NOE ASSIGNMENT ALGORITHM USING PROTEIN BACKBONE DETERMINED FROM RESIDUAL DIPOLAR COUPLINGS AND ROTAMER PATTERNS." Comput Syst Bioinformatics Conf 2008 (2008): 169-181.
PMID
19122773
Source
pubmed
Published In
Computational systems bioinformatics / Life Sciences Society. Computational Systems Bioinformatics Conference
Volume
2008
Publish Date
2008
Start Page
169
End Page
181

A Hausdorff-based NOE assignment algorithm using protein backbone determined from residual dipolar couplings and rotamer patterns.

High-throughput structure determination based on solution Nuclear Magnetic Resonance (NMR) spectroscopy plays an important role in structural genomics. One of the main bottlenecks in NMR structure determination is the interpretation of NMR data to obtain a sufficient number of accurate distance restraints by assigning nuclear Overhauser effect (NOE) spectral peaks to pairs of protons. The difficulty in automated NOE assignment mainly lies in the ambiguities arising both from the resonance degeneracy of chemical shifts and from the uncertainty due to experimental errors in NOE peak positions. In this paper we present a novel NOE assignment algorithm, called HAusdorff-based NOE Assignment (HANA), that starts with a high-resolution protein backbone computed using only two residual dipolar couplings (RDCs) per residue, employs a Hausdorff-based pattern matching technique to deduce similarity between experimental and back-computed NOE spectra for each rotamer from a statistically diverse library, and drives the selection of optimal position-specific rotamers for filtering ambiguous NOE assignments. Our algorithm runs in time O(tn3 + tn log t), where t is the maximum number of rotamers per residue and n is the size of the protein. Application of our algorithm on biological NMR data for three proteins, namely, human ubiquitin, the zinc finger domain of the human DNA Y-polymerase Eta (pol eta) and the human Set2-Rpb1 interacting domain (hSRI) demonstrates that our algorithm overcomes spectral noise to achieve more than 90% assignment accuracy. Additionally, the final structures calculated using our automated NOE assignments have backbone RMSD < 1.7 A and all-heavy-atom RMSD < 2.5 A from reference structures that were determined either by X-ray crystallography or traditional NMR approaches. These results show that our NOE assignment algorithm can be successfully applied to protein NMR spectra to obtain high-quality structures.

Authors
Zeng, J; Tripathy, C; Zhou, P; Donald, BR
MLA Citation
Zeng, J, Tripathy, C, Zhou, P, and Donald, BR. "A Hausdorff-based NOE assignment algorithm using protein backbone determined from residual dipolar couplings and rotamer patterns." Comput Syst Bioinformatics Conf 7 (2008): 169-181.
PMID
19642278
Source
pubmed
Published In
Computational systems bioinformatics / Life Sciences Society. Computational Systems Bioinformatics Conference
Volume
7
Publish Date
2008
Start Page
169
End Page
181

Structure of the deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent inhibition and specificity in ligand binding.

The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising antibiotic target. CHIR-090, the most potent LpxC inhibitor discovered to date, displays two-step time-dependent inhibition and kills a wide range of Gram-negative pathogens as effectively as ciprofloxacin or tobramycin. In this study, we report the solution structure of the LpxC-CHIR-090 complex. CHIR-090 exploits conserved features of LpxC that are critical for catalysis, including the hydrophobic passage and essential active-site residues. CHIR-090 is adjacent to, but does not occupy, the UDP-binding pocket of LpxC, suggesting that a fragment-based approach may facilitate further optimization of LpxC inhibitors. Additionally, we identified key residues in the Insert II hydrophobic passage that modulate time-dependent inhibition and CHIR-090 resistance. CHIR-090 shares a similar, although previously unrecognized, chemical scaffold with other small-molecule antibiotics such as L-161,240 targeting LpxC, and provides a template for understanding the binding mode of these inhibitors. Consistent with this model, we provide evidence that L-161,240 also occupies the hydrophobic passage.

Authors
Barb, AW; Jiang, L; Raetz, CRH; Zhou, P
MLA Citation
Barb, AW, Jiang, L, Raetz, CRH, and Zhou, P. "Structure of the deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent inhibition and specificity in ligand binding." Proc Natl Acad Sci U S A 104.47 (November 20, 2007): 18433-18438.
PMID
18025458
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
47
Publish Date
2007
Start Page
18433
End Page
18438
DOI
10.1073/pnas.0709412104

Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin.

The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here, we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisting of 12 residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the alpha 3 helix and the central beta-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76(Ub) carboxylate group either by a peptide or isopeptide bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as "sensors" of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes.

Authors
Pai, M-T; Tzeng, S-R; Kovacs, JJ; Keaton, MA; Li, SS-C; Yao, T-P; Zhou, P
MLA Citation
Pai, M-T, Tzeng, S-R, Kovacs, JJ, Keaton, MA, Li, SS-C, Yao, T-P, and Zhou, P. "Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin." J Mol Biol 370.2 (July 6, 2007): 290-302.
PMID
17512543
Source
pubmed
Published In
Journal of Molecular Biology
Volume
370
Issue
2
Publish Date
2007
Start Page
290
End Page
302
DOI
10.1016/j.jmb.2007.04.015

Inhibition of lipid A biosynthesis as the primary mechanism of CHIR-090 antibiotic activity in Escherichia coli.

The deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against Pseudomonas aeruginosa and Escherichia coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of E. coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1, and k6 = 0.18 min-1. CHIR-090 at low nanomolar levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including P. aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 microM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 microg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway.

Authors
Barb, AW; McClerren, AL; Snehelatha, K; Reynolds, CM; Zhou, P; Raetz, CRH
MLA Citation
Barb, AW, McClerren, AL, Snehelatha, K, Reynolds, CM, Zhou, P, and Raetz, CRH. "Inhibition of lipid A biosynthesis as the primary mechanism of CHIR-090 antibiotic activity in Escherichia coli." Biochemistry 46.12 (March 27, 2007): 3793-3802.
PMID
17335290
Source
pubmed
Published In
Biochemistry
Volume
46
Issue
12
Publish Date
2007
Start Page
3793
End Page
3802
DOI
10.1021/bi6025165

Structure of the ubiquitin-binding zinc finger domain of human DNA Y-polymerase eta.

The ubiquitin-binding zinc finger (UBZ) domain of human DNA Y-family polymerase (pol) eta is important in the recruitment of the polymerase to the stalled replication machinery in translesion synthesis. Here, we report the solution structure of the pol eta UBZ domain and its interaction with ubiquitin. We show that the UBZ domain adopts a classical C(2)H(2) zinc-finger structure characterized by a betabetaalpha fold. Nuclear magnetic resonance titration maps the binding interfaces between UBZ and ubiquitin to the alpha-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single alpha-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol eta UBZ domain represents a novel member of the C(2)H(2) zinc finger family that interacts with ubiquitin to regulate translesion synthesis.

Authors
Bomar, MG; Pai, M-T; Tzeng, S-R; Li, SS-C; Zhou, P
MLA Citation
Bomar, MG, Pai, M-T, Tzeng, S-R, Li, SS-C, and Zhou, P. "Structure of the ubiquitin-binding zinc finger domain of human DNA Y-polymerase eta." EMBO Rep 8.3 (March 2007): 247-251.
PMID
17304240
Source
pubmed
Published In
EMBO Reports
Volume
8
Issue
3
Publish Date
2007
Start Page
247
End Page
251
DOI
10.1038/sj.embor.7400901

Sampling of the NMR time domain along concentric rings.

We present a novel approach to sampling the NMR time domain, whereby the sampling points are aligned on concentric rings, which we term concentric ring sampling (CRS). Radial sampling constitutes a special case of CRS where each ring has the same number of points and the same relative orientation. We derive theoretically that the most efficient CRS approach is to place progressively more points on rings of larger radius, with the number of points growing linearly with the radius, a method that we call linearly increasing CRS (LCRS). For cases of significant undersampling to reduce measurement time, a randomized LCRS (RLCRS) is also described. A theoretical treatment of these approaches is provided, including an assessment of artifacts and sensitivity. The analytical treatment of sensitivity also addresses the sensitivity of radially sampled data processed by Fourier transform. Optimized CRS approaches are found to produce artifact-free spectra of the same resolution as Cartesian sampling, for the same measurement time. Additionally, optimized approaches consistently yield fewer and smaller artifacts than radial sampling, and have a sensitivity equal to Cartesian and better than radial sampling. We demonstrate the method using numerical simulations, as well as a 3D HNCO experiment on protein G B1 domain.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "Sampling of the NMR time domain along concentric rings." J Magn Reson 184.2 (February 2007): 207-221.
PMID
17070715
Source
pubmed
Published In
Journal of Magnetic Resonance
Volume
184
Issue
2
Publish Date
2007
Start Page
207
End Page
221
DOI
10.1016/j.jmr.2006.10.002

Structure of the deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent inhibition and specificity in ligand binding (Proceedings of National Academy of Sciences of the United States of America (November 20, 2007) 104, 47, (18433-18438) DOI:10.1073/pnas0709412104))

Authors
Barb, AW; Jiang, L; Raetz, CRH; Zhou, P
MLA Citation
Barb, AW, Jiang, L, Raetz, CRH, and Zhou, P. "Structure of the deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent inhibition and specificity in ligand binding (Proceedings of National Academy of Sciences of the United States of America (November 20, 2007) 104, 47, (18433-18438) DOI:10.1073/pnas0709412104))." Proceedings of the National Academy of Sciences of the United States of America 104.52 (2007): 21020--.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
52
Publish Date
2007
Start Page
21020-
DOI
10.1073/pnas.0711056105

The ADAM10 prodomain is a specific inhibitor of ADAM10 proteolytic activity and inhibits cellular shedding events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-α- converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a Ki of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Moss, ML; Bomar, M; Liu, Q; Sage, H; Dempsey, P; Lenhart, PM; Gillispie, PA; Stoeck, A; Wildeboer, D; Bartsch, JW; Palmisano, R; Zhou, P
MLA Citation
Moss, ML, Bomar, M, Liu, Q, Sage, H, Dempsey, P, Lenhart, PM, Gillispie, PA, Stoeck, A, Wildeboer, D, Bartsch, JW, Palmisano, R, and Zhou, P. "The ADAM10 prodomain is a specific inhibitor of ADAM10 proteolytic activity and inhibits cellular shedding events." Journal of Biological Chemistry 282.49 (2007): 35712-35721.
PMID
17895248
Source
scival
Published In
The Journal of biological chemistry
Volume
282
Issue
49
Publish Date
2007
Start Page
35712
End Page
35721
DOI
10.1074/jbc.M703231200

Drosophila PIWI associates with chromatin and interacts directly with HP1a

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of PIWI. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. We also demonstrate that PIWI, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the PIWI-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome. © 2007 by Cold Spring Harbor Laboratory Press.

Authors
Brower-Toland, B; Findley, SD; Jiang, L; Liu, L; Yin, H; Dus, M; Zhou, P; Elgin, SCR; Lin, H
MLA Citation
Brower-Toland, B, Findley, SD, Jiang, L, Liu, L, Yin, H, Dus, M, Zhou, P, Elgin, SCR, and Lin, H. "Drosophila PIWI associates with chromatin and interacts directly with HP1a." Genes and Development 21.18 (2007): 2300-2311.
PMID
17875665
Source
scival
Published In
Genes & development
Volume
21
Issue
18
Publish Date
2007
Start Page
2300
End Page
2311
DOI
10.1101/gad.1564307

The low affinity IgE receptor (CD23) is cleaved by the metalloproteinase ADAM10

The low affinity IgE receptor, FcεRII (CD23), is both a positive and negative regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species (sCD23) is an important regulator of the function of CD23 and may be an important therapeutic target for the control of allergy and inflammation. We have characterized the catalytic activity ofADAM(a disintegrin and metalloproteinase) 10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in the CD23 backbone. Tissue inhibitors of metalloproteinases and a specific prodomain-based inhibitor of ADAM10 perturb the release of endogenously produced CD23 from human leukemia cell lines as well as primary cultures of human B-cells. Expression of a mutant metalloproteinase-deficient construct of ADAM10 partially inhibited the production of sCD23. Similarly, small inhibitory RNA knockdown of ADAM10 partially inhibited CD23 release and resulted in the accumulation of the membrane-bound form of CD23 on the cells. ADAM10 contributes to CD23 shedding and thus could be considered a potential therapeutic target for the treatment of allergic disease.

Authors
Lemieux, GA; Blumenkron, F; Yeung, N; Zhou, P; Williams, J; Grammer, AC; Petrovich, R; Lipsky, PE; Moss, ML; Werb, Z
MLA Citation
Lemieux, GA, Blumenkron, F, Yeung, N, Zhou, P, Williams, J, Grammer, AC, Petrovich, R, Lipsky, PE, Moss, ML, and Werb, Z. "The low affinity IgE receptor (CD23) is cleaved by the metalloproteinase ADAM10." Journal of Biological Chemistry 282.20 (2007): 14836-14844.
PMID
17389606
Source
scival
Published In
The Journal of biological chemistry
Volume
282
Issue
20
Publish Date
2007
Start Page
14836
End Page
14844
DOI
10.1074/jbc.M608414200

Polar Fourier transforms of radially sampled NMR data.

Radial sampling of the NMR time domain has recently been introduced to speed up data collection significantly. Here, we show that radially sampled data can be processed directly using Fourier transforms in polar coordinates. We present a comprehensive theoretical analysis of the discrete polar Fourier transform, and derive the consequences of its application to radially sampled data using linear response theory. With adequate sampling, the resulting spectrum using a polar Fourier transform is indistinguishable from conventionally processed spectra with Cartesian sampling. In the case of undersampling in azimuth--the condition that provides significant savings in measurement time-the correct spectrum is still produced, but with limited distortion of the baseline away from the peaks, taking the form of a summation of high-order Bessel functions. Finally, we describe an intrinsic connection between the polar Fourier transform and the filtered backprojection method that has recently been introduced to process projection-reconstruction NOESY data. Direct polar Fourier transformation holds great potential for producing quantitatively accurate spectra from radially sampled NMR data.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "Polar Fourier transforms of radially sampled NMR data." J Magn Reson 182.1 (September 2006): 84-95.
PMID
16820311
Source
pubmed
Published In
Journal of Magnetic Resonance
Volume
182
Issue
1
Publish Date
2006
Start Page
84
End Page
95
DOI
10.1016/j.jmr.2006.06.016

A 'just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity.

Among the suite of commonly used backbone experiments, HNCACO presents an unresolved sensitivity limitation due to fast 13CO transverse relaxation and passive 13Calpha-13Cbeta coupling. Here, we present a high-sensitivity 'just-in-time' (JIT) HN(CA)CO pulse sequence that uniformly refocuses 13Calpha-13Cbeta coupling while collecting 13CO shifts in real time. Sensitivity comparisons of the 3-D JIT HN(CA)CO, a CT-HMQC-based control, and a HSQC-based control with selective 13Calpha inversion pulses were performed using a 2H/13C/15N labeled sample of the 29 kDa HCA II protein at 15 degrees C. The JIT experiment shows a 42% signal enhancement over the CT-HMQC-based experiment. Compared to the HSQC-based experiment, the JIT experiment is 16% less sensitive for residues experiencing proper 13Calpha refocusing and13Calpha-13Cbeta decoupling. However, for the remaining residues, the JIT spectrum shows a 106% average sensitivity gain over the HSQC-based experiment. The high-sensitivity JIT HNCACO experiment should be particularly beneficial for studies of large proteins to provide 13CO resonance information regardless of residue type.

Authors
Werner-Allen, JW; Jiang, L; Zhou, P
MLA Citation
Werner-Allen, JW, Jiang, L, and Zhou, P. "A 'just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity." J Magn Reson 181.1 (July 2006): 177-180.
PMID
16644250
Source
pubmed
Published In
Journal of Magnetic Resonance
Volume
181
Issue
1
Publish Date
2006
Start Page
177
End Page
180
DOI
10.1016/j.jmr.2006.04.001

Structure and CTD phosphorylation pattern-binding specificity of the SRI domain of Set2

Authors
Greenleaf, AL; Phatnani, HP; Li, M; Zhou, P
MLA Citation
Greenleaf, AL, Phatnani, HP, Li, M, and Zhou, P. "Structure and CTD phosphorylation pattern-binding specificity of the SRI domain of Set2." March 6, 2006.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
20
Issue
4
Publish Date
2006
Start Page
A467
End Page
A467

Evaluating the quality of NMR structures by local density of protons.

Evaluating the quality of experimentally determined protein structural models is an essential step toward identifying potential errors and guiding further structural refinement. Herein, we report the use of proton local density as a sensitive measure to assess the quality of nuclear magnetic resonance (NMR) structures. Using 256 high-resolution crystal structures with protons added and optimized, we show that the local density of different proton types display distinct distributions. These distributions can be characterized by statistical moments and are used to establish local density Z-scores for evaluating both global and local packing for individual protons. Analysis of 546 crystal structures at various resolutions shows that the local density Z-scores increase as the structural resolution decreases and correlate well with the ClashScore (Word et al. J Mol Biol 1999;285(4):1711-1733) generated by all atom contact analysis. Local density Z-scores for NMR structures exhibit a significantly wider range of values than for X-ray structures and demonstrate a combination of potentially problematic inflation and compression. Water-refined NMR structures show improved packing quality. Our analysis of a high-quality structural ensemble of ubiquitin refined against order parameters shows proton density distributions that correlate nearly perfectly with our standards derived from crystal structures, further validating our approach. We present an automated analysis and visualization tool for proton packing to evaluate the quality of NMR structures.

Authors
Ban, Y-EA; Rudolph, J; Zhou, P; Edelsbrunner, H
MLA Citation
Ban, Y-EA, Rudolph, J, Zhou, P, and Edelsbrunner, H. "Evaluating the quality of NMR structures by local density of protons." Proteins 62.4 (March 1, 2006): 852-864.
PMID
16342274
Source
pubmed
Published In
Proteins: Structure, Function and Bioinformatics
Volume
62
Issue
4
Publish Date
2006
Start Page
852
End Page
864
DOI
10.1002/prot.20811

PR-CALC: a program for the reconstruction of NMR spectra from projections.

Projection-reconstruction NMR (PR-NMR) has attracted growing attention as a method for collecting multidimensional NMR data rapidly. The PR-NMR procedure involves measuring lower-dimensional projections of a higher-dimensional spectrum, which are then used for the mathematical reconstruction of the full spectrum. We describe here the program PR-CALC, for the reconstruction of NMR spectra from projection data. This program implements a number of reconstruction algorithms, highly optimized to achieve maximal performance, and manages the reconstruction process automatically, producing either full spectra or subsets, such as regions or slices, as requested. The ability to obtain subsets allows large spectra to be analyzed by reconstructing and examining only those subsets containing peaks, offering considerable savings in processing time and storage space. PR-CALC is straightforward to use, and integrates directly into the conventional pipeline for data processing and analysis. It was written in standard C+ + and should run on any platform. The organization is flexible, and permits easy extension of capabilities, as well as reuse in new software. PR-CALC should facilitate the widespread utilization of PR-NMR in biomedical research.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "PR-CALC: a program for the reconstruction of NMR spectra from projections." J Biomol NMR 34.3 (March 2006): 179-195.
PMID
16604426
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
34
Issue
3
Publish Date
2006
Start Page
179
End Page
195
DOI
10.1007/s10858-006-0020-z

NMR assignment of the SRI domain of human Set2/HYPB.

Authors
Li, M; Phatnani, HP; Greenleaf, AL; Zhou, P
MLA Citation
Li, M, Phatnani, HP, Greenleaf, AL, and Zhou, P. "NMR assignment of the SRI domain of human Set2/HYPB." J Biomol NMR 36 Suppl 1 (2006): 5-. (Letter)
PMID
16435090
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
36 Suppl 1
Publish Date
2006
Start Page
5
DOI
10.1007/s10858-005-4690-8

Solution structure of the Set2-Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1.

The phosphorylation state of the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II changes as polymerase transcribes a gene, and the distinct forms of the phospho-CTD (PCTD) recruit different nuclear factors to elongating polymerase. The Set2 histone methyltransferase from yeast was recently shown to bind the PCTD of elongating RNA polymerase II by means of a novel domain termed the Set2-Rpb1 interacting (SRI) domain. Here, we report the solution structure of the SRI domain in human Set2 (hSRI domain), which adopts a left-turned three-helix bundle distinctly different from other structurally characterized PCTD-interacting domains. NMR titration experiments mapped the binding surface of the hSRI domain to helices 1 and 2, and Biacore binding studies showed that the domain binds preferably to [Ser-2 + Ser-5]-phosphorylated CTD peptides containing two or more heptad repeats. Point-mutagenesis studies identified five residues critical for PCTD binding. In view of the differential effects of these point mutations on binding to different CTD phosphopeptides, we propose a model for the hSRI domain interaction with the PCTD.

Authors
Li, M; Phatnani, HP; Guan, Z; Sage, H; Greenleaf, AL; Zhou, P
MLA Citation
Li, M, Phatnani, HP, Guan, Z, Sage, H, Greenleaf, AL, and Zhou, P. "Solution structure of the Set2-Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1." Proc Natl Acad Sci U S A 102.49 (December 6, 2005): 17636-17641.
PMID
16314571
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
49
Publish Date
2005
Start Page
17636
End Page
17641
DOI
10.1073/pnas.0506350102

Filtered backprojection for the reconstruction of a high-resolution (4,2)D CH3-NH NOESY spectrum on a 29 kDa protein.

Projection-reconstruction (PR) NMR enables rapid collection of multidimensional NMR data. NOESY represents a particularly difficult challenge for currently existing reconstruction algorithms, as it requires the quantitative reconstruction of an unknown number of peaks, at full sensitivity. We have demonstrated the successful application of PR-NMR to NOESY by determining the 4D methyl/amide NOESY spectrum of a 29 kDa protein, human carbonic anhydrase II, from 2D projections, using filtered backprojection for reconstruction. Compared with a 3D control spectrum, all expected peaks were faithfully reconstructed, with correct volumes and with no artifacts. Filtered backprojection thus provides a way to obtain high-resolution 4D NOESY data in the time required for conventional 3D data collection.

Authors
Coggins, BE; Venters, RA; Zhou, P
MLA Citation
Coggins, BE, Venters, RA, and Zhou, P. "Filtered backprojection for the reconstruction of a high-resolution (4,2)D CH3-NH NOESY spectrum on a 29 kDa protein." J Am Chem Soc 127.33 (August 24, 2005): 11562-11563.
PMID
16104707
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
127
Issue
33
Publish Date
2005
Start Page
11562
End Page
11563
DOI
10.1021/ja053110k

Rapid assignment of protein side chain resonances using projection-reconstruction of (4,3)D HC(CCO)NH and intra-HC(C)NH experiments.

The reconstruction of higher-dimensional NMR spectra from projections can provide significant savings in instrument time. Here, we demonstrate its application to the (4,3)D HC(CCO)NH and intra-HC(C)NH experiments. The latter experiment contains a novel intra-residue filter element, which selectively correlates the side chain resonances with the corresponding intra-residue amide resonances. Compared to the conventional HC(C)NH experiment, the intra-HC(C)NH experiment reduces the spectral complexity and thus the minimum number of projections required for artifact-free reconstruction by half. The use of the projection-reconstruction technique allows rapid data collection and unambiguous assignment of aliphatic side chain nuclei at high resolution.

Authors
Jiang, L; Coggins, BE; Zhou, P
MLA Citation
Jiang, L, Coggins, BE, and Zhou, P. "Rapid assignment of protein side chain resonances using projection-reconstruction of (4,3)D HC(CCO)NH and intra-HC(C)NH experiments." J Magn Reson 175.1 (July 2005): 170-176.
PMID
15949755
Source
pubmed
Published In
Journal of Magnetic Resonance
Volume
175
Issue
1
Publish Date
2005
Start Page
170
End Page
176
DOI
10.1016/j.jmr.2005.03.014

(4,2)D Projection--reconstruction experiments for protein backbone assignment: application to human carbonic anhydrase II and calbindin D(28K).

Projection-reconstruction NMR experiments have been shown to significantly reduce the acquisition time required to obtain protein backbone assignment data. To date, this concept has only been applied to smaller (15)N/(13)C-labeled proteins. Here, we show that projection-reconstruction NMR techniques can be extended to larger protonated and perdeuterated proteins. We present a suite of (4,2)D triple-resonance experiments for protein backbone assignment and a Hybrid Backprojection/Lower-Value algorithm for reconstructing data with relatively weak signal-to-noise ratios. In addition, we propose a sampling theorem and discuss its implication on the choice of projection angles. We demonstrate the efficacy of this approach using the 29 kDa protein, human carbonic anhydrase II and the 30 kDa protein, calbindin D(28K).

Authors
Venters, RA; Coggins, BE; Kojetin, D; Cavanagh, J; Zhou, P
MLA Citation
Venters, RA, Coggins, BE, Kojetin, D, Cavanagh, J, and Zhou, P. "(4,2)D Projection--reconstruction experiments for protein backbone assignment: application to human carbonic anhydrase II and calbindin D(28K)." J Am Chem Soc 127.24 (June 22, 2005): 8785-8795.
PMID
15954785
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
127
Issue
24
Publish Date
2005
Start Page
8785
End Page
8795
DOI
10.1021/ja0509580

Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway.

The first committed step of lipid A biosynthesis in Gram-negative bacteria is catalyzed by the zinc-dependent hydrolase LpxC that removes an acetate from the nitrogen at the 2' '-position of UDP-3-O-acyl-N-acetylglucosamine. Recent structural characterization by both NMR and X-ray crystallography provides many important details about the active site environment of LpxC from Aquifex aeolicus, a heat-stable orthologue that displays 32% sequence identity to LpxC from Escherichia coli. The detailed reaction mechanism and specific roles of active site residues for LpxC from A. aeolicus are further analyzed here. The pH dependencies of k(cat)/K(M) and k(cat) for the deacetylation of the substrate UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc are both bell-shaped. The ascending acidic limb (pK(1)) was fitted to 6.1 +/- 0.2 for k(cat) and 5.7 +/- 0.2 for k(cat)/K(M). The descending basic limb (pK(2)) was fitted to 8.0 +/- 0.2 for k(cat) and 8.4 +/- 0.2 for k(cat)/K(M). The pH dependence of the E73A mutant exhibits loss of the acidic limb, and the mutant retains only 0.15% activity versus the wild type. The pH dependencies of the other active site mutants H253A, K227A, H253A/K227A, and D234N remain bell-shaped, although their significantly lower activities (0.25%, 0.05%, 0.007%, and 0.57%, respectively) suggest that they contribute significantly to catalysis. Our cumulative data support a mechanism for LpxC wherein Glu73 serves as the general base for deprotonation and activation of the zinc-bound water.

Authors
McClerren, AL; Zhou, P; Guan, Z; Raetz, CRH; Rudolph, J
MLA Citation
McClerren, AL, Zhou, P, Guan, Z, Raetz, CRH, and Rudolph, J. "Kinetic analysis of the zinc-dependent deacetylase in the lipid A biosynthetic pathway." Biochemistry 44.4 (February 1, 2005): 1106-1113.
PMID
15667204
Source
pubmed
Published In
Biochemistry
Volume
44
Issue
4
Publish Date
2005
Start Page
1106
End Page
1113
DOI
10.1021/bi048001h

Refined solution structure of the LpxC-TU-514 complex and pKa analysis of an active site histidine: insights into the mechanism and inhibitor design.

Lipopolysaccharide, the major constituent of the outer monolayer of the outer membrane of Gram-negative bacteria, is anchored into the membrane through the hydrophobic moiety lipid A, a hexaacylated disaccharide. The zinc-dependent metalloamidase UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the second and committed step in the biosynthesis of lipid A. LpxC shows no homology to mammalian metalloamidases and is essential for cell viability, making it an important target for the development of novel antibacterial compounds. Recent NMR and X-ray studies of the LpxC from Aquifex aeolicus have provided the first structural information about this family of proteins. Insight into the catalytic mechanism and the design of effective inhibitors could be facilitated by more detailed structural and biochemical studies that define substrate-protein interactions and the roles of specific residues in the active site. Here, we report the synthesis of the (13)C-labeled substrate-analogue inhibitor TU-514, and the subsequent refinement of the solution structure of the A. aeolicus LpxC-TU-514 complex using residual dipolar couplings. We also reevaluate the catalytic role of an active site histidine, H253, on the basis of both its pK(a) as determined by NMR titration and pH-dependent kinetic analyses. These results provide a structural basis for the design of more potent LpxC inhibitors than those that are currently available.

Authors
Coggins, BE; McClerren, AL; Jiang, L; Li, X; Rudolph, J; Hindsgaul, O; Raetz, CRH; Zhou, P
MLA Citation
Coggins, BE, McClerren, AL, Jiang, L, Li, X, Rudolph, J, Hindsgaul, O, Raetz, CRH, and Zhou, P. "Refined solution structure of the LpxC-TU-514 complex and pKa analysis of an active site histidine: insights into the mechanism and inhibitor design." Biochemistry 44.4 (February 1, 2005): 1114-1126.
PMID
15667205
Source
pubmed
Published In
Biochemistry
Volume
44
Issue
4
Publish Date
2005
Start Page
1114
End Page
1126
DOI
10.1021/bi047820z

A methylation-dependent electrostatic switch controls DNA repair and transcriptional activation by E. coli Ada

The transcriptional activity of many sequence-specific DNA binding proteins is directly regulated by posttranslational covalent modification. Although this form of regulation was first described nearly two decades ago, it remains poorly understood at a mechanistic level. The prototype for a transcription factor controlled by posttranslational modification is E. coli Ada protein, a chemosensor that both repairs methylation damage in DNA and coordinates the resistance response to genotoxic methylating agents. Ada repairs methyl phosphotriester lesions in DNA by transferring the aberrant methyl group to one of its own cysteine residues; this site-specific methylation enhances tremendously the DNA binding activity of the protein, thereby enabling it to activate a methylation-resistance regulon. Here, we report solution and X-ray structures of the Cys-methylated chemosensor domain of Ada bound to DNA. The structures reveal that both phosphotriester repair and methylation-dependent transcriptional activation function through a zinc- and methylation-dependent electrostatic switch. Copyright ©2005 by Elsevier Inc.

Authors
He, C; Hus, J-C; Li, JS; Zhou, P; Norman, DPG; Dötsch, V; Wei, H; Gross, JD; Lane, WS; Wagner, G; Verdine, GL
MLA Citation
He, C, Hus, J-C, Li, JS, Zhou, P, Norman, DPG, Dötsch, V, Wei, H, Gross, JD, Lane, WS, Wagner, G, and Verdine, GL. "A methylation-dependent electrostatic switch controls DNA repair and transcriptional activation by E. coli Ada." Molecular Cell 20.1 (2005): 117-129.
PMID
16209950
Source
scival
Published In
Molecular Cell
Volume
20
Issue
1
Publish Date
2005
Start Page
117
End Page
129
DOI
10.1016/j.molcel.2005.08.013

Mechanistic studies of the deacetylase LpxC, a novel target for drug design in gram-negative bacteria

Authors
McClerren, A; Rudolph, J; Zhou, P; Raetz, CRH
MLA Citation
McClerren, A, Rudolph, J, Zhou, P, and Raetz, CRH. "Mechanistic studies of the deacetylase LpxC, a novel target for drug design in gram-negative bacteria." May 14, 2004.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
18
Issue
8
Publish Date
2004
Start Page
C54
End Page
C54

Generalized reconstruction of n-D NMR spectra from multiple projections: application to the 5-D HACACONH spectrum of protein G B1 domain.

Reconstructing multidimensional NMR spectra from 2-D projections significantly reduces the time needed for data collection over conventional methodology. Here, we provide a generalization of the projection-reconstruction process to spectra of arbitrary dimensionality, using a concept of coordinate rotation to produce explicit expressions for reconstruction. These expressions allow one to reconstruct subsets of the higher dimensionality space without producing the full spectrum, permitting convenient analysis of the data. We demonstrate the effectiveness of these procedures in the reconstruction of the 5-D HACACONH spectrum of protein G B1 domain from 12 2-D projections collected in five experiments. We further demonstrate that the base spectra of GFT-NMR are equivalent to projections of the 5-D spectrum at fixed angles.

Authors
Coggins, BE; Venters, RA; Zhou, P
MLA Citation
Coggins, BE, Venters, RA, and Zhou, P. "Generalized reconstruction of n-D NMR spectra from multiple projections: application to the 5-D HACACONH spectrum of protein G B1 domain." J Am Chem Soc 126.4 (February 4, 2004): 1000-1001.
PMID
14746450
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
126
Issue
4
Publish Date
2004
Start Page
1000
End Page
1001
DOI
10.1021/ja039430q

Assignment of the 1H, 13C and 15N resonances of the LpxC deacetylase from Aquifex aeolicus in complex with the substrate-analog inhibitor TU-514.

Authors
Coggins, BE; Li, X; Hindsgaul, O; Raetz, CRH; Zhou, P
MLA Citation
Coggins, BE, Li, X, Hindsgaul, O, Raetz, CRH, and Zhou, P. "Assignment of the 1H, 13C and 15N resonances of the LpxC deacetylase from Aquifex aeolicus in complex with the substrate-analog inhibitor TU-514." J Biomol NMR 28.2 (February 2004): 201-202. (Letter)
PMID
14755169
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
28
Issue
2
Publish Date
2004
Start Page
201
End Page
202
DOI
10.1023/B:JNMR.0000013817.29493.87

Structure of the LpxC deacetylase with a bound substrate-analog inhibitor.

The zinc-dependent UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the first committed step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharide (LPS) that constitutes the outermost monolayer of Gram-negative bacteria. As LpxC is crucial for the survival of Gram-negative organisms and has no sequence homology to known mammalian deacetylases or amidases, it is an excellent target for the design of new antibiotics. The solution structure of LpxC from Aquifex aeolicus in complex with a substrate-analog inhibitor, TU-514, reveals a novel alpha/beta fold, a unique zinc-binding motif and a hydrophobic passage that captures the acyl chain of the inhibitor. On the basis of biochemical and structural studies, we propose a catalytic mechanism for LpxC, suggest a model for substrate binding and provide evidence that mobility and dynamics in structural motifs close to the active site have key roles in the capture of the substrate.

Authors
Coggins, BE; Li, X; McClerren, AL; Hindsgaul, O; Raetz, CRH; Zhou, P
MLA Citation
Coggins, BE, Li, X, McClerren, AL, Hindsgaul, O, Raetz, CRH, and Zhou, P. "Structure of the LpxC deacetylase with a bound substrate-analog inhibitor." Nat Struct Biol 10.8 (August 2003): 645-651.
PMID
12833153
Source
pubmed
Published In
Nature Structural Biology
Volume
10
Issue
8
Publish Date
2003
Start Page
645
End Page
651
DOI
10.1038/nsb948

PACES: Protein sequential assignment by computer-assisted exhaustive search.

A crucial step in determining solution structures of proteins using nuclear magnetic resonance (NMR) spectroscopy is the process of sequential assignment, which correlates backbone resonances to corresponding residues in the primary sequence of a protein, today, typically using data from triple-resonance NMR experiments. Although the development of automated approaches for sequential assignment has greatly facilitated this process, the performance of these programs is usually less satisfactory for large proteins, especially in the cases of missing connectivity or severe chemical shift degeneracy. Here, we report the development of a novel computer-assisted method for sequential assignment, using an algorithm that conducts an exhaustive search of all spin systems both for establishing sequential connectivities and then for assignment. By running the program iteratively with user intervention after each cycle, ambiguities in the assignments can be eliminated efficiently and backbone resonances can be assigned rapidly. The efficiency and robustness of this approach have been tested with 27 proteins of sizes varying from 76 amino acids to 723 amino acids, and with data of varying qualities, using experimental data for three proteins, and published assignments modified with simulated noise for the other 24. The complexity of sequential assignment with regard to the size of the protein, the completeness of NMR data sets, and the uncertainty in resonance positions has been examined.

Authors
Coggins, BE; Zhou, P
MLA Citation
Coggins, BE, and Zhou, P. "PACES: Protein sequential assignment by computer-assisted exhaustive search." J Biomol NMR 26.2 (June 2003): 93-111.
PMID
12766406
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
26
Issue
2
Publish Date
2003
Start Page
93
End Page
111

Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates.

Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the approximately 70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of approximately 10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only approximately 9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination.

Authors
Blackshear, PJ; Lai, WS; Kennington, EA; Brewer, G; Wilson, GM; Guan, X; Zhou, P
MLA Citation
Blackshear, PJ, Lai, WS, Kennington, EA, Brewer, G, Wilson, GM, Guan, X, and Zhou, P. "Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates." J Biol Chem 278.22 (May 30, 2003): 19947-19955.
PMID
12639954
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
22
Publish Date
2003
Start Page
19947
End Page
19955
DOI
10.1074/jbc.M301290200

A novel approach for characterizing protein ligand complexes: molecular basis for specificity of small-molecule Bcl-2 inhibitors.

The increasing diversity of small molecule libraries has been an important source for the development of new drugs and, more recently, for unraveling the mechanisms of cellular events-a process termed chemical genetics.(1) Unfortunately, the majority of currently available compounds are mechanism-based enzyme inhibitors, whereas most of cellular activity regulation proceeds on the level of protein-protein interactions. Hence, the development of small molecule inhibitors of protein-protein interactions is important. When screening compound libraries, low-micromolar inhibitors of protein interactions can be routinely found. The enhancement of affinities and rationalization of the binding mechanism require structural information about the protein-ligand complexes. Crystallization of low-affinity complexes is difficult, and their NMR analysis suffers from exchange broadening, which limits the number of obtainable intermolecular constraints. Here we present a novel method of ligand validation and optimization, which is based on the combination of structural and computational approaches. We successfully used this method to analyze the basis for structure-activity relationships of previously selected (2) small molecule inhibitors of the antiapoptotic protein Bcl-xL and identified new members of this inhibitor family.

Authors
Lugovskoy, AA; Degterev, AI; Fahmy, AF; Zhou, P; Gross, JD; Yuan, J; Wagner, G
MLA Citation
Lugovskoy, AA, Degterev, AI, Fahmy, AF, Zhou, P, Gross, JD, Yuan, J, and Wagner, G. "A novel approach for characterizing protein ligand complexes: molecular basis for specificity of small-molecule Bcl-2 inhibitors." J Am Chem Soc 124.7 (February 20, 2002): 1234-1240.
PMID
11841292
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
124
Issue
7
Publish Date
2002
Start Page
1234
End Page
1240

Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45.

Apoptotic DNA fragmentation is mediated by a caspase-activated DNA fragmentation factor (DFF)40. Expression and folding of DFF40 require the presence of DFF45, which also acts as a nuclease inhibitor before DFF40 activation by execution caspases. The N-terminal domains (NTDs) of both proteins are homologous, and their interaction plays a key role in the proper functioning of this two-component system. Here we report that the NTD of DFF45 alone is unstructured in solution, and its folding is induced upon binding to DFF40 NTD. Therefore, folding of both proteins regulates the formation of the DFF40/DFF45 complex. The solution structure of the heterodimeric complex between NTDs of DFF40 and DFF45 reported here shows that the mutual chaperoning includes the formation of an extensive network of intermolecular interactions that bury a hydrophobic cluster inside the interface, surrounded by intermolecular salt bridges.

Authors
Zhou, P; Lugovskoy, AA; McCarty, JS; Li, P; Wagner, G
MLA Citation
Zhou, P, Lugovskoy, AA, McCarty, JS, Li, P, and Wagner, G. "Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45." Proc Natl Acad Sci U S A 98.11 (May 22, 2001): 6051-6055.
PMID
11371636
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
11
Publish Date
2001
Start Page
6051
End Page
6055
DOI
10.1073/pnas.111145098

A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins.

Protein-fusion constructs have been used with great success for enhancing expression of soluble recombinant protein and as tags for affinity purification. Unfortunately the most popular tags, such as GST and MBP, are large, which hinders direct NMR studies of the fusion proteins. Cleavage of the fusion proteins often re-introduces problems with solubility and stability. Here we describe the use of N-terminally fused protein G (B1 domain) as a non-cleavable solubility-enhancement tag (SET) for structure determination of a dimeric protein complex. The SET enhances the solubility and stability of the fusion product dramatically while not interacting directly with the protein of interest. This approach can be used for structural characterization of poorly behaving protein systems, and would be especially useful for structural genomics studies.

Authors
Zhou, P; Lugovskoy, AA; Wagner, G
MLA Citation
Zhou, P, Lugovskoy, AA, and Wagner, G. "A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins." J Biomol NMR 20.1 (May 2001): 11-14.
PMID
11430750
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
20
Issue
1
Publish Date
2001
Start Page
11
End Page
14

Solution structure of the CIDE-N domain of CIDE-B and a model for CIDE-N/CIDE-N interactions in the DNA fragmentation pathway of apoptosis.

Apoptotic DNA fragmentation and chromatin condensation are mediated by the caspase-activated DFF40/ CAD nuclease, which is chaperoned and inhibited by DFF45/ICAD. CIDE proteins share a homologous regulatory CIDE-N domain with DFF40/CAD and DFF45/ ICAD. Here we report the solution structure of CIDE-N of human CIDE-B. We show that the CIDE-N of CIDE-B interacts with CIDE-N domains of both DFF40 and DFF45. The binding epitopes are similar and map to a highly charged bipolar surface region of CIDE-B. Furthermore, we demonstrate that the CIDE-N of CIDE-B regulates enzymatic activity of the DFF40/ DFF45 complex in vitro. Based on these results and mutagenesis data, we propose a model for the CIDE-N/ CIDE-N complex and discuss the role of this novel bipolar interaction in mediating downstream events of apoptosis.

Authors
Lugovskoy, AA; Zhou, P; Chou, JJ; McCarty, JS; Li, P; Wagner, G
MLA Citation
Lugovskoy, AA, Zhou, P, Chou, JJ, McCarty, JS, Li, P, and Wagner, G. "Solution structure of the CIDE-N domain of CIDE-B and a model for CIDE-N/CIDE-N interactions in the DNA fragmentation pathway of apoptosis." Cell 99.7 (December 23, 1999): 747-755.
PMID
10619428
Source
pubmed
Published In
Cell
Volume
99
Issue
7
Publish Date
1999
Start Page
747
End Page
755

Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adaptor/caspase interaction.

Direct recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD/CARD (Caspase Recruitment Domain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic alpha-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD/CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD/CARD interaction between Apaf-1 and caspase-9.

Authors
Zhou, P; Chou, J; Olea, RS; Yuan, J; Wagner, G
MLA Citation
Zhou, P, Chou, J, Olea, RS, Yuan, J, and Wagner, G. "Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adaptor/caspase interaction." Proc Natl Acad Sci U S A 96.20 (September 28, 1999): 11265-11270.
PMID
10500165
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
20
Publish Date
1999
Start Page
11265
End Page
11270

Solution structure of the core NFATC1/DNA complex.

The nuclear factor of the activated T cell (NFAT) family of transcription factors regulates cytokine gene expression by binding to the promoter/enhancer regions of antigen-responsive genes, usually in cooperation with heterologous DNA-binding partners. Here we report the solution structure of the binary complex formed between the core DNA-binding domain of human NFATC1 and the ARRE2 DNA site from the interleukin-2 promoter. The structure reveals that DNA binding induces the folding of key structural elements that are required for both sequence-specific recognition and the establishment of cooperative protein-protein contacts. The orientation of the NFAT DNA-binding domain observed in the binary NFATC1-DBD*/ DNA complex is distinct from that seen in the ternary NFATC2/AP-1/DNA complex, suggesting that the domain reorients upon formation of a cooperative transcriptional complex.

Authors
Zhou, P; Sun, LJ; Dötsch, V; Wagner, G; Verdine, GL
MLA Citation
Zhou, P, Sun, LJ, Dötsch, V, Wagner, G, and Verdine, GL. "Solution structure of the core NFATC1/DNA complex." Cell 92.5 (March 6, 1998): 687-696.
PMID
9506523
Source
pubmed
Published In
Cell
Volume
92
Issue
5
Publish Date
1998
Start Page
687
End Page
696

Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc.

Transcription factors of the NFAT family regulate the production of effector proteins that coordinate the immune response. The immunosuppressive drugs FK506 and cyclosporin A (CsA) act by blocking a Ca2+-mediated signalling pathway leading to NFAT. Although FK506 and CsA have enabled human organs to be transplanted routinely, the toxic side-effects of these drugs limit their usage. This toxicity might be absent in antagonists that target NFAT directly. As a first step in the structure-based search for NFAT antagonists, we now report the identification and solution structure of a 20K domain of NFATc (NFATc-DBD) that is both necessary and sufficient to bind DNA and activate transcription cooperatively. Although the overall fold of the NFATc DNA-binding domain is related to that of NF-kappaB p50 (refs 2, 3), the two proteins use significantly different strategies for DNA recognition. On the basis of these results, we present a model for the cooperative complex formed between NFAT and the mitogenic transcription factor AP-1 on the interleukin-2 enhancer.

Authors
Wolfe, SA; Zhou, P; Dötsch, V; Chen, L; You, A; Ho, SN; Crabtree, GR; Wagner, G; Verdine, GL
MLA Citation
Wolfe, SA, Zhou, P, Dötsch, V, Chen, L, You, A, Ho, SN, Crabtree, GR, Wagner, G, and Verdine, GL. "Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc." Nature 385.6612 (January 9, 1997): 172-176.
PMID
8990122
Source
pubmed
Published In
Nature
Volume
385
Issue
6612
Publish Date
1997
Start Page
172
End Page
176
DOI
10.1038/385172a0
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