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Beese, Lorena Sue

Overview:

Overview. The broad goal of our research is to understand biological processes in atomic detail. A multi-disciplinary strategy is employed using macromolecular X-ray crystallography to determine high resolution, three-dimensional images of proteins and appropriate complexes. The structural information is combined with biochemical, genetic, and computational analyses to address questions central to cancer biology. In addition, this approach may facilitate the development new therapeutic agents for the treatment cancer and other diseases.

Signal transduction. Numerous signal transduction proteins, including members of the Ras GTPase superfamily, require posttranslational attachment of an isoprenoid lipid group for proper function. A major focus of the lab is on understanding the structural enzymology of the protein prenyltransferase family of lipid modifying enzymes. We have determined high resolution, three-dimensional structures of human protein farnesyltransferase (FTase), an enzyme that is considered a promising anticancer drug target. Inhibitors of FTase cause tumor regression in animals and are currently being evaluated in clinical trials for the treatment of human cancer. Subsequently, we have determined a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the structural determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes.

Structure based drug design. More than 300 patent applications have been made so far for prenyltransferase inhibitors, and at least six are currently in clinical trials. The laboratory is using structural differences among protein prenyltransferases to develop highly specific inhibitors. Our laboratory has determined the mechanism of action of peptidomemetic inhibitors that showed tumor regression in animal studies and are currently investigating chemotherapreutics used in clinical trials for treatment of human cancer. The structures facilitate the design new drugs targeting the prenyltransferase enzyme family. Inhibitors that specifically target the prenyltransferase of pathogens such as Plasmodium falciparum or Trypanosoma brucei may lead to improved treatments for diseases like maleria.

DNA Mismatch Repair. The laboratory is investigating protein DNA assemblies involved in human mismatch repair. Mismatch repair is essential for maintaining genomic stability of all organisms. Defects in genes involved in mismatch repair lead to elevated mutation rates, and in the case of humans confer a strong predisposition to tumorigenesis. Currently, experiments are focused on determining structures of protein-DNA assemblies involved in the initiation of the human mismatch repair reaction.

DNA replication. A major focus is on understanding the molecular mechanisms of DNA replication. We have determined high-resolution crystal structures of DNA polymerases with DNA primer-templates that capture different stages of the synthesis reaction. Of particular interest is the Bacillus DNA polymerase that retains its ability for processive, accurate DNA synthesis in the crystal. We are using this polymerase as a model system to study molecular mechanisms of DNA mispair incorporation and action of carcinogens that can lead to mutations.

Observing Enzymes in Action. A common theme of the laboratory is the study of enzyme mechanisms at near atomic resolution by determining three-dimensional structures that represent stages along the reaction pathway. Structural information is combined with biochemical, biophysical, and computational analyses to understand how enzymes function. An exciting new direction arises from the observation that one of the DNA polymerases retains catalytic activity in the crystal (see above). Currently, we are developing methodology to study the phosphoryl-transfer reaction using time-resolved crystallography. Our goal is to observe DNA synthesis in real time. Ultimately, this many enable the dynamic process of accurate DNA replication to be viewed and constrasted with replication under mutagenic conditions.

Positions:

James B. Duke Professor of Medicine

Biochemistry
School of Medicine

Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1984

Ph.D. — Brandeis University

News:

Nobel Prize in Chemistry awarded to 3 scientists for DNA repair discovery

October 08, 2015 — NPR’s “All Things Considered”

Grants:

Enzymology Of Eukaryotic Mismatch Repair

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
January 01, 1991
End Date
February 28, 2019

Structural Cell Biology of DNA Repair Machines Sub: Mismatch Repair Interactions

Administered By
Biochemistry
AwardedBy
Ernest Orlando Lawrence Berkeley National Laboratory
Role
Principal Investigator
Start Date
December 13, 2001
End Date
August 31, 2016

Structure and Mechanism of Protein Prenyltransferases

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 24, 1995
End Date
February 29, 2016

Structural biology of human DNA mismatch repair machinery

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2009
End Date
August 31, 2012

Structure And Mechanism Of Protein Prenyl Transferases

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 24, 1995
End Date
April 30, 1999

Structure And Mechanism Of Protein Prenyltransferases

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 24, 1995
End Date
April 30, 1999

Rotating Anode X-Ray Generator & Image Plate System

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
August 15, 1996
End Date
August 14, 1997
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Awards:

Members/ Foreign Associates. National Academy of Science.

Type
National
Awarded By
National Academy of Science
Date
January 01, 2009

Scholars. Searle Scholars.

Type
National
Awarded By
Searle Scholars
Date
January 01, 1994

Publications:

The Closing Mechanism of DNA Polymerase I at Atomic Resolution.

DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl.

Authors
Miller, BR; Beese, LS; Parish, CA; Wu, EY
MLA Citation
Miller, BR, Beese, LS, Parish, CA, and Wu, EY. "The Closing Mechanism of DNA Polymerase I at Atomic Resolution." Structure (London, England : 1993) 23.9 (September 2015): 1609-1620.
PMID
26211612
Source
epmc
Published In
Structure
Volume
23
Issue
9
Publish Date
2015
Start Page
1609
End Page
1620
DOI
10.1016/j.str.2015.06.016

Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues.

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme.

Authors
Wang, Y-C; Dozier, JK; Beese, LS; Distefano, MD
MLA Citation
Wang, Y-C, Dozier, JK, Beese, LS, and Distefano, MD. "Rapid analysis of protein farnesyltransferase substrate specificity using peptide libraries and isoprenoid diphosphate analogues." ACS chemical biology 9.8 (August 2014): 1726-1735.
PMID
24841702
Source
epmc
Published In
ACS Chemical Biology
Volume
9
Issue
8
Publish Date
2014
Start Page
1726
End Page
1735
DOI
10.1021/cb5002312

Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design.

Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high-resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide substrate, corresponding to successive steps in ordered substrate binding, revealed that the second substrate-binding step is accompanied by motions of a loop in the catalytic site. Re-examination of other FTase structures showed that this motion is conserved. The substrate- and product-binding clefts in the AfFTase active site are wider than in human FTase (hFTase). Widening is a consequence of small shifts in the α-helices that comprise the majority of the FTase structure, which in turn arise from sequence variation in the hydrophobic core of the protein. These structural effects are key features that distinguish fungal FTases from hFTase. Their variation results in differences in steady-state enzyme kinetics and inhibitor interactions and presents opportunities for developing selective anti-fungal drugs by exploiting size differences in the active sites. We illustrate the latter by comparing the interaction of ED5 and Tipifarnib with hFTase and AfFTase. In AfFTase, the wider groove enables ED5 to bind in the presence of FPP, whereas in hFTase it binds only in the absence of substrate. Tipifarnib binds similarly to both enzymes but makes less extensive contacts in AfFTase with consequently weaker binding.

Authors
Mabanglo, MF; Hast, MA; Lubock, NB; Hellinga, HW; Beese, LS
MLA Citation
Mabanglo, MF, Hast, MA, Lubock, NB, Hellinga, HW, and Beese, LS. "Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design." Protein Sci 23.3 (March 2014): 289-301.
PMID
24347326
Source
pubmed
Published In
Protein Science
Volume
23
Issue
3
Publish Date
2014
Start Page
289
End Page
301
DOI
10.1002/pro.2411

Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation.

We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using a cell-free protein engineering automation methodology that bypasses gene cloning, we iteratively constructed, produced, and assayed hundreds of mutations in binding-site residues to identify improvements in Clomeleon, a first-generation, suboptimal sensor. Structural analysis revealed that these improvements involve halide contacts and distant side chain rearrangements. The development of optogenetic sensors that respond to neural activity enables cellular tracking of neural activity using optical, rather than electrophysiological, signals. Construction of such sensors using in vitro protein engineering establishes a powerful approach for developing new probes for brain imaging.

Authors
Grimley, JS; Li, L; Wang, W; Wen, L; Beese, LS; Hellinga, HW; Augustine, GJ
MLA Citation
Grimley, JS, Li, L, Wang, W, Wen, L, Beese, LS, Hellinga, HW, and Augustine, GJ. "Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation." J Neurosci 33.41 (October 9, 2013): 16297-16309.
PMID
24107961
Source
pubmed
Published In
The Journal of neuroscience : the official journal of the Society for Neuroscience
Volume
33
Issue
41
Publish Date
2013
Start Page
16297
End Page
16309
DOI
10.1523/JNEUROSCI.4616-11.2013

Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides.

In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2'-hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base pair discrimination.

Authors
Wang, W; Wu, EY; Hellinga, HW; Beese, LS
MLA Citation
Wang, W, Wu, EY, Hellinga, HW, and Beese, LS. "Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides." J Biol Chem 287.34 (August 17, 2012): 28215-28226.
PMID
22648417
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
34
Publish Date
2012
Start Page
28215
End Page
28226
DOI
10.1074/jbc.M112.366609

Covalent protein-oligonucleotide conjugates by copper-free click reaction.

Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates-where the connection between the two components is at a defined location in both the protein and the ODN-under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were 'clicked' to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.

Authors
Khatwani, SL; Kang, JS; Mullen, DG; Hast, MA; Beese, LS; Distefano, MD; Taton, TA
MLA Citation
Khatwani, SL, Kang, JS, Mullen, DG, Hast, MA, Beese, LS, Distefano, MD, and Taton, TA. "Covalent protein-oligonucleotide conjugates by copper-free click reaction." Bioorg Med Chem 20.14 (July 15, 2012): 4532-4539.
PMID
22682299
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
20
Issue
14
Publish Date
2012
Start Page
4532
End Page
4539
DOI
10.1016/j.bmc.2012.05.017

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis.

Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C • A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

Authors
Wang, W; Hellinga, HW; Beese, LS
MLA Citation
Wang, W, Hellinga, HW, and Beese, LS. "Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis." Proc Natl Acad Sci U S A 108.43 (October 25, 2011): 17644-17648.
PMID
22006298
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
108
Issue
43
Publish Date
2011
Start Page
17644
End Page
17648
DOI
10.1073/pnas.1114496108

Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

Authors
Hast, MA; Nichols, CB; Armstrong, SM; Kelly, SM; Hellinga, HW; Alspaugh, JA; Beese, LS
MLA Citation
Hast, MA, Nichols, CB, Armstrong, SM, Kelly, SM, Hellinga, HW, Alspaugh, JA, and Beese, LS. "Structures of Cryptococcus neoformans protein farnesyltransferase reveal strategies for developing inhibitors that target fungal pathogens." J Biol Chem 286.40 (October 7, 2011): 35149-35162.
PMID
21816822
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
40
Publish Date
2011
Start Page
35149
End Page
35162
DOI
10.1074/jbc.M111.250506

Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ.

MutSβ is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2-MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSβ. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSβ and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.

Authors
Tseng, Q; Orans, J; Hast, MA; Iyer, RR; Changela, A; Modrich, PL; Beese, LS
MLA Citation
Tseng, Q, Orans, J, Hast, MA, Iyer, RR, Changela, A, Modrich, PL, and Beese, LS. "Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ." Acta Crystallogr Sect F Struct Biol Cryst Commun 67.Pt 8 (August 1, 2011): 947-952.
PMID
21821902
Source
pubmed
Published In
Acta Crystallographica Section F
Volume
67
Issue
Pt 8
Publish Date
2011
Start Page
947
End Page
952
DOI
10.1107/S1744309111019300

The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism.

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established "open" and "closed" states. In this "ajar" conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation.

Authors
Wu, EY; Beese, LS
MLA Citation
Wu, EY, and Beese, LS. "The structure of a high fidelity DNA polymerase bound to a mismatched nucleotide reveals an "ajar" intermediate conformation in the nucleotide selection mechanism." J Biol Chem 286.22 (June 3, 2011): 19758-19767.
PMID
21454515
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
22
Publish Date
2011
Start Page
19758
End Page
19767
DOI
10.1074/jbc.M110.191130

Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family.

Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5' structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5' ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.

Authors
Orans, J; McSweeney, EA; Iyer, RR; Hast, MA; Hellinga, HW; Modrich, P; Beese, LS
MLA Citation
Orans, J, McSweeney, EA, Iyer, RR, Hast, MA, Hellinga, HW, Modrich, P, and Beese, LS. "Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family." Cell 145.2 (April 15, 2011): 212-223.
PMID
21496642
Source
pubmed
Published In
Cell
Volume
145
Issue
2
Publish Date
2011
Start Page
212
End Page
223
DOI
10.1016/j.cell.2011.03.005

Structural Biochemistry of CaaX Protein Prenyltransferases

Protein prenylation is a posttranslational lipid modification required for proper function by over 100 proteins in the eukaryotic cell. A family of structurally related protein prenyltransferase enzymes carry out this reaction: protein farnesyltransferase (FTase), protein geranylgeranyltransferase-I (GGTase-I), and Rab geranylgeranyltransferase (GGTase-II or Rab GGTase). This chapter concerns the structural biology of Ca 1a 2X protein prenyltransferases (FTase and GGTase-I). These enzymes recognize a well-defined C-terminal motif on substrate proteins: cysteine (C), followed by two generally aliphatic amino acids (aa) and a variable (X) residue. FTase and GGTase-I catalyze the addition of a 15-carbon or 20-carbon isoprenoid lipid, respectively. FTase and GGTase-I have been shown to be important targets for the development of cancer chemotherapeutics because prenylated signal transduction proteins play significant roles in oncogenesis. More recently, it has been demonstrated that protein prenyltransferases also show promise as drug targets for treating a variety of infectious diseases caused by fungi and protozoans. We review the structural features of the enzymatic reaction cycle, and how these relate to the mechanisms of inhibition by small molecules. We also review recent structural studies of human pathogen protein prenyltransferases, and how this information can be used for developing species-specific prenyltransferase inhibitors to treat infectious diseases. © 2011 Elsevier Inc.

Authors
Hast, MA; Beese, LS
MLA Citation
Hast, MA, and Beese, LS. "Structural Biochemistry of CaaX Protein Prenyltransferases." Enzymes 29 (2011): 235-257.
Source
scival
Published In
Enzymes
Volume
29
Publish Date
2011
Start Page
235
End Page
257
DOI
10.1016/B978-0-12-381339-8.00013-5

Structure-based design and synthesis of potent, ethylenediamine-based, mammalian farnesyltransferase inhibitors as anticancer agents.

A potent class of anticancer, human farnesyltransferase (hFTase) inhibitors has been identified by "piggy-backing" on potent, antimalarial inhibitors of Plasmodium falciparum farnesyltransferase (PfFTase). On the basis of a 4-fold substituted ethylenediamine scaffold, the inhibitors are structurally simple and readily derivatized, facilitating the extensive structure-activity relationship (SAR) study reported herein. Our most potent inhibitor is compound 1f, which exhibited an in vitro hFTase IC(50) value of 25 nM and a whole cell H-Ras processing IC(50) value of 90 nM. Moreover, it is noteworthy that several of our inhibitors proved highly selective for hFTase (up to 333-fold) over the related prenyltransferase enzyme geranylgeranyltransferase-I (GGTase-I). A crystal structure of inhibitor 1a co-crystallized with farnesyl pyrophosphate (FPP) in the active site of rat FTase illustrates that the para-benzonitrile moiety of 1a is stabilized by a π-π stacking interaction with the Y361β residue, suggesting a structural explanation for the observed importance of this component of our inhibitors.

Authors
Fletcher, S; Keaney, EP; Cummings, CG; Blaskovich, MA; Hast, MA; Glenn, MP; Chang, S-Y; Bucher, CJ; Floyd, RJ; Katt, WP; Gelb, MH; Van Voorhis, WC; Beese, LS; Sebti, SM; Hamilton, AD
MLA Citation
Fletcher, S, Keaney, EP, Cummings, CG, Blaskovich, MA, Hast, MA, Glenn, MP, Chang, S-Y, Bucher, CJ, Floyd, RJ, Katt, WP, Gelb, MH, Van Voorhis, WC, Beese, LS, Sebti, SM, and Hamilton, AD. "Structure-based design and synthesis of potent, ethylenediamine-based, mammalian farnesyltransferase inhibitors as anticancer agents." J Med Chem 53.19 (October 14, 2010): 6867-6888.
Website
http://hdl.handle.net/10161/4056
PMID
20822181
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
53
Issue
19
Publish Date
2010
Start Page
6867
End Page
6888
DOI
10.1021/jm1001748

MutLalpha and proliferating cell nuclear antigen share binding sites on MutSbeta.

MutSbeta (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSbeta interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSbeta-PCNA complex formation occurs with an affinity of approximately 0.1 microM and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSbeta is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSbeta not only abolishes PCNA binding, but unlike MutSalpha, also dramatically attenuates MutSbeta-MutLalpha interaction, MutLalpha endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLalpha for binding to MutSbeta, an effect that is blocked by the cell cycle regulator p21(CIP1). We propose that MutSbeta-MutLalpha interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSbeta- and MutSalpha-initiated repair events differ in fundamental ways.

Authors
Iyer, RR; Pluciennik, A; Genschel, J; Tsai, M-S; Beese, LS; Modrich, P
MLA Citation
Iyer, RR, Pluciennik, A, Genschel, J, Tsai, M-S, Beese, LS, and Modrich, P. "MutLalpha and proliferating cell nuclear antigen share binding sites on MutSbeta." J Biol Chem 285.15 (April 9, 2010): 11730-11739.
PMID
20154325
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
15
Publish Date
2010
Start Page
11730
End Page
11739
DOI
10.1074/jbc.M110.104125

The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis.

High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1 helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.

Authors
Golosov, AA; Warren, JJ; Beese, LS; Karplus, M
MLA Citation
Golosov, AA, Warren, JJ, Beese, LS, and Karplus, M. "The mechanism of the translocation step in DNA replication by DNA polymerase I: a computer simulation analysis." Structure 18.1 (January 13, 2010): 83-93.
PMID
20152155
Source
pubmed
Published In
Structure
Volume
18
Issue
1
Publish Date
2010
Start Page
83
End Page
93
DOI
10.1016/j.str.2009.10.014

Structural analysis of semi-specific oligosaccharide recognition by a cellulose-binding protein of thermotoga maritima reveals adaptations for functional diversification of the oligopeptide periplasmic binding protein fold.

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of beta(1-->4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.

Authors
Cuneo, MJ; Beese, LS; Hellinga, HW
MLA Citation
Cuneo, MJ, Beese, LS, and Hellinga, HW. "Structural analysis of semi-specific oligosaccharide recognition by a cellulose-binding protein of thermotoga maritima reveals adaptations for functional diversification of the oligopeptide periplasmic binding protein fold." J Biol Chem 284.48 (November 27, 2009): 33217-33223.
PMID
19801540
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
48
Publish Date
2009
Start Page
33217
End Page
33223
DOI
10.1074/jbc.M109.041624

Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.

We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N(2), N-3, and N(6) of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects. Second, it dramatically increased the rate of misincorporation of the resulting dNTP analogues, with alterations in either N-1 or N(6) having the most severe impacts. Adding N(2) to dNTPs containing the bases adenine and purine increased the degree of polymerization opposite T but also tremendously increased the degree of misincorporation opposite A, C, and G. Thus, BF uses N-1, N(2), N-3, and N(6) of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation. Comparing how BF discriminates between right and wrong dNTPs with both B family polymerases and low-fidelity polymerases indicates that BF has chosen a unique solution vis-a-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.

Authors
Trostler, M; Delier, A; Beckman, J; Urban, M; Patro, JN; Spratt, TE; Beese, LS; Kuchta, RD
MLA Citation
Trostler, M, Delier, A, Beckman, J, Urban, M, Patro, JN, Spratt, TE, Beese, LS, and Kuchta, RD. "Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus." Biochemistry 48.21 (June 2, 2009): 4633-4641.
PMID
19348507
Source
pubmed
Published In
Biochemistry
Volume
48
Issue
21
Publish Date
2009
Start Page
4633
End Page
4641
DOI
10.1021/bi900104n

Structural adaptations that modulate monosaccharide, disaccharide, and trisaccharide specificities in periplasmic maltose-binding proteins.

Periplasmic binding proteins comprise a superfamily that is present in archaea, prokaryotes, and eukaryotes. Periplasmic binding protein ligand-binding sites have diversified to bind a wide variety of ligands. Characterization of the structural mechanisms by which functional adaptation occurs is key to understanding the evolution of this important protein superfamily. Here we present the structure and ligand-binding properties of a maltotriose-binding protein identified from the Thermus thermophilus genome sequence. We found that this receptor has a high affinity for the trisaccharide maltotriose (K(d)<1 microM) but little affinity for disaccharides that are transported by a paralogous maltose transport operon present in T. thermophilus. Comparison of this structure to other proteins that adopt the maltose-binding protein fold but bind monosaccharides, disaccharides, or trisaccharides reveals the presence of four subsites that bind individual glucose ring units. Two loops and three helical segments encode adaptations that control the presence of each subsite by steric blocking or hydrogen bonding. We provide a model in which the energetics of long-range conformational equilibria controls subsite occupancy and ligand binding.

Authors
Cuneo, MJ; Changela, A; Beese, LS; Hellinga, HW
MLA Citation
Cuneo, MJ, Changela, A, Beese, LS, and Hellinga, HW. "Structural adaptations that modulate monosaccharide, disaccharide, and trisaccharide specificities in periplasmic maltose-binding proteins." J Mol Biol 389.1 (May 29, 2009): 157-166.
PMID
19361522
Source
pubmed
Published In
Journal of Molecular Biology
Volume
389
Issue
1
Publish Date
2009
Start Page
157
End Page
166
DOI
10.1016/j.jmb.2009.04.008

Structural basis for binding and selectivity of antimalarial and anticancer ethylenediamine inhibitors to protein farnesyltransferase.

Protein farnesyltransferase (FTase) catalyzes an essential posttranslational lipid modification of more than 60 proteins involved in intracellular signal transduction networks. FTase inhibitors have emerged as a significant target for development of anticancer therapeutics and, more recently, for the treatment of parasitic diseases caused by protozoan pathogens, including malaria (Plasmodium falciparum). We present the X-ray crystallographic structures of complexes of mammalian FTase with five inhibitors based on an ethylenediamine scaffold, two of which exhibit over 1000-fold selective inhibition of P. falciparum FTase. These structures reveal the dominant determinants in both the inhibitor and enzyme that control binding and selectivity. Comparison to a homology model constructed for the P. falciparum FTase suggests opportunities for further improving selectivity of a new generation of antimalarial inhibitors.

Authors
Hast, MA; Fletcher, S; Cummings, CG; Pusateri, EE; Blaskovich, MA; Rivas, K; Gelb, MH; Van Voorhis, WC; Sebti, SM; Hamilton, AD; Beese, LS
MLA Citation
Hast, MA, Fletcher, S, Cummings, CG, Pusateri, EE, Blaskovich, MA, Rivas, K, Gelb, MH, Van Voorhis, WC, Sebti, SM, Hamilton, AD, and Beese, LS. "Structural basis for binding and selectivity of antimalarial and anticancer ethylenediamine inhibitors to protein farnesyltransferase." Chem Biol 16.2 (February 27, 2009): 181-192.
PMID
19246009
Source
pubmed
Published In
Chemistry and Biology
Volume
16
Issue
2
Publish Date
2009
Start Page
181
End Page
192
DOI
10.1016/j.chembiol.2009.01.014

Structural analysis of a periplasmic binding protein in the tripartite ATP-independent transporter family reveals a tetrameric assembly that may have a role in ligand transport.

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Authors
Cuneo, MJ; Changela, A; Miklos, AE; Beese, LS; Krueger, JK; Hellinga, HW
MLA Citation
Cuneo, MJ, Changela, A, Miklos, AE, Beese, LS, Krueger, JK, and Hellinga, HW. "Structural analysis of a periplasmic binding protein in the tripartite ATP-independent transporter family reveals a tetrameric assembly that may have a role in ligand transport." J Biol Chem 283.47 (November 21, 2008): 32812-32820.
PMID
18723845
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
47
Publish Date
2008
Start Page
32812
End Page
32820
DOI
10.1074/jbc.M803595200

Ligand-induced conformational changes in a thermophilic ribose-binding protein.

BACKGROUND: Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. RESULTS: Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (app Tm value is 108 degrees C) than the mesophilic Escherichia coli homolog (ecRBP) (app Tm value is 56 degrees C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. CONCLUSION: Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.

Authors
Cuneo, MJ; Beese, LS; Hellinga, HW
MLA Citation
Cuneo, MJ, Beese, LS, and Hellinga, HW. "Ligand-induced conformational changes in a thermophilic ribose-binding protein. (Published online)" BMC Struct Biol 8 (November 19, 2008): 50-.
PMID
19019243
Source
pubmed
Published In
BMC Structural Biology
Volume
8
Publish Date
2008
Start Page
50
DOI
10.1186/1472-6807-8-50

Structure of protein geranylgeranyltransferase-I from the human pathogen Candida albicans complexed with a lipid substrate.

Protein geranylgeranyltransferase-I (GGTase-I) catalyzes the transfer of a 20-carbon isoprenoid lipid to the sulfur of a cysteine residue located near the C terminus of numerous cellular proteins, including members of the Rho superfamily of small GTPases and other essential signal transduction proteins. In humans, GGTase-I and the homologous protein farnesyltransferase (FTase) are targets of anticancer therapeutics because of the role small GTPases play in oncogenesis. Protein prenyltransferases are also essential for many fungal and protozoan pathogens that infect humans, and have therefore become important targets for treating infectious diseases. Candida albicans, a causative agent of systemic fungal infections in immunocompromised individuals, is one pathogen for which protein prenylation is essential for survival. Here we present the crystal structure of GGTase-I from C. albicans (CaGGTase-I) in complex with its cognate lipid substrate, geranylgeranylpyrophosphate. This structure provides a high-resolution picture of a non-mammalian protein prenyltransferase. There are significant variations between species in critical areas of the active site, including the isoprenoid-binding pocket, as well as the putative product exit groove. These differences indicate the regions where specific protein prenyltransferase inhibitors with antifungal activity can be designed.

Authors
Hast, MA; Beese, LS
MLA Citation
Hast, MA, and Beese, LS. "Structure of protein geranylgeranyltransferase-I from the human pathogen Candida albicans complexed with a lipid substrate." J Biol Chem 283.46 (November 14, 2008): 31933-31940.
PMID
18713740
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
46
Publish Date
2008
Start Page
31933
End Page
31940
DOI
10.1074/jbc.M805330200

Caged protein prenyltransferase substrates: tools for understanding protein prenylation.

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.

Authors
DeGraw, AJ; Hast, MA; Xu, J; Mullen, D; Beese, LS; Barany, G; Distefano, MD
MLA Citation
DeGraw, AJ, Hast, MA, Xu, J, Mullen, D, Beese, LS, Barany, G, and Distefano, MD. "Caged protein prenyltransferase substrates: tools for understanding protein prenylation." Chem Biol Drug Des 72.3 (September 2008): 171-181.
PMID
18844669
Source
pubmed
Published In
Chemical Biology & Drug Design
Volume
72
Issue
3
Publish Date
2008
Start Page
171
End Page
181
DOI
10.1111/j.1747-0285.2008.00698.x

The MutSalpha-proliferating cell nuclear antigen interaction in human DNA mismatch repair.

We have examined the interaction parameters, conformation, and functional significance of the human MutSalpha(.) proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a K(D) of 0.7 microm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSalpha for a mismatch, and mismatch-bound MutSalpha binds PCNA. Small angle x-ray scattering studies have established the molecular parameters of the complex, which are consistent with an elongated conformation in which the two proteins associate in an end-to-end fashion in a manner that does not involve an extended unstructured tether, as has been proposed for yeast MutSalpha and PCNA ( Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell 26, 565-578 ). MutSalpha variants lacking the PCNA interaction motif are functional in 3'- or 5'-directed mismatch-provoked excision, but display a partial defect in 5'-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSalpha PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSalpha-dependent mismatch repair.

Authors
Iyer, RR; Pohlhaus, TJ; Chen, S; Hura, GL; Dzantiev, L; Beese, LS; Modrich, P
MLA Citation
Iyer, RR, Pohlhaus, TJ, Chen, S, Hura, GL, Dzantiev, L, Beese, LS, and Modrich, P. "The MutSalpha-proliferating cell nuclear antigen interaction in human DNA mismatch repair." J Biol Chem 283.19 (May 9, 2008): 13310-13319.
PMID
18326858
Source
pubmed
Published In
The Journal of biological chemistry
Volume
283
Issue
19
Publish Date
2008
Start Page
13310
End Page
13319
DOI
10.1074/jbc.M800606200

Dissecting the differences between the alpha and beta anomers of the oxidative DNA lesion FaPydG.

The oxidative DNA lesion, FaPydG rapidly anomerizes to form a mixture of the alpha and beta anomer. To investigate the mutagenic potential of both forms, we prepared stabilized bioisosteric analogues of both configurational isomers and incorporated them into oligonucleotides. These were subsequently used for thermodynamic melting-point studies and for primer-extension experiments. While the beta compound, in agreement with earlier data, prefers cytidine as the pairing partner, the alpha compound is not able form a stable base pair with any natural base. In primer-extension studies with the high-fidelity polymerase Bst Pol I, the polymerase was able to read through the lesion. The beta compound showed no strong mutagenic potential. The alpha compound, in contrast, strongly destabilized DNA duplexes and also blocked all of the tested DNA polymerases, including two low-fidelity polymerases of the Y-family.

Authors
Büsch, F; Pieck, JC; Ober, M; Gierlich, J; Hsu, GW; Beese, LS; Carell, T
MLA Citation
Büsch, F, Pieck, JC, Ober, M, Gierlich, J, Hsu, GW, Beese, LS, and Carell, T. "Dissecting the differences between the alpha and beta anomers of the oxidative DNA lesion FaPydG." Chemistry 14.7 (2008): 2125-2132.
PMID
18196510
Source
pubmed
Published In
Chemistry - A European Journal
Volume
14
Issue
7
Publish Date
2008
Start Page
2125
End Page
2132
DOI
10.1002/chem.200701373

Dissecting the differences between the alpha and beta anomers of the oxidative DNA lesion FaPydG.

The oxidative DNA lesion, FaPydG rapidly anomerizes to form a mixture of the alpha and beta anomer. To investigate the mutagenic potential of both forms, we prepared stabilized bioisosteric analogues of both configurational isomers and incorporated them into oligonucleotides. These were subsequently used for thermodynamic melting-point studies and for primer-extension experiments. While the beta compound, in agreement with earlier data, prefers cytidine as the pairing partner, the alpha compound is not able form a stable base pair with any natural base. In primer-extension studies with the high-fidelity polymerase Bst Pol I, the polymerase was able to read through the lesion. The beta compound showed no strong mutagenic potential. The alpha compound, in contrast, strongly destabilized DNA duplexes and also blocked all of the tested DNA polymerases, including two low-fidelity polymerases of the Y-family.

Authors
Büsch, F; Pieck, JC; Ober, M; Gierlich, J; Hsu, GW; Beese, LS; Carell, T
MLA Citation
Büsch, F, Pieck, JC, Ober, M, Gierlich, J, Hsu, GW, Beese, LS, and Carell, T. "Dissecting the differences between the alpha and beta anomers of the oxidative DNA lesion FaPydG." Chemistry (Weinheim an der Bergstrasse, Germany) 14.7 (2008): 2125-2132.
Source
scival
Published In
Chemistry - A European Journal
Volume
14
Issue
7
Publish Date
2008
Start Page
2125
End Page
2132
DOI
10.1002/chem.200701373

Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein.

We report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose-binding protein obtained from Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic DNA and overexpressed in Escherichia coli, the identity of its cognate sugar was confirmed, ligand binding was studied, and the structure of its glucose complex was solved to 1.7 Angstrom resolution by X-ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 +/- 1 degrees C and 144 +/- 2 degrees C in the absence and presence of 1 mM glucose, respectively). A series of fluorescent conjugates was constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site-specific mutagenesis at positions predicted to be responsive to ligand-induced conformational changes based on the structure. These conjugates were screened to identify engineered tmGBPs that function as reagentless fluorescent glucose biosensors. The Y13C*Cy5 conjugate is bright, gives a large response to glucose over concentration ranges appropriate for in vivo monitoring of blood glucose levels (1-30 mM), and can be immobilized in an orientation-specific manner in microtiter plates to give a reversible response to glucose. The immobilized protein retains its response after long-term storage at room temperature.

Authors
Tian, Y; Cuneo, MJ; Changela, A; Höcker, B; Beese, LS; Hellinga, HW
MLA Citation
Tian, Y, Cuneo, MJ, Changela, A, Höcker, B, Beese, LS, and Hellinga, HW. "Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein." Protein Sci 16.10 (October 2007): 2240-2250.
PMID
17766373
Source
pubmed
Published In
Protein Science
Volume
16
Issue
10
Publish Date
2007
Start Page
2240
End Page
2250
DOI
10.1110/ps.072969407

Structure of the human MutSalpha DNA lesion recognition complex.

Mismatch repair (MMR) ensures the fidelity of DNA replication, initiates the cellular response to certain classes of DNA damage, and has been implicated in the generation of immune diversity. Each of these functions depends on MutSalpha (MSH2*MSH6 heterodimer). Inactivation of this protein complex is responsible for tumor development in about half of known hereditary nonpolyposis colorectal cancer kindreds and also occurs in sporadic tumors in a variety of tissues. Here, we describe a series of crystal structures of human MutSalpha bound to different DNA substrates, each known to elicit one of the diverse biological responses of the MMR pathway. All lesions are recognized in a similar manner, indicating that diversity of MutSalpha-dependent responses to DNA lesions is generated in events downstream of this lesion recognition step. This study also allows rigorous mapping of cancer-causing mutations and furthermore suggests structural pathways for allosteric communication between different regions within the heterodimer.

Authors
Warren, JJ; Pohlhaus, TJ; Changela, A; Iyer, RR; Modrich, PL; Beese, LS
MLA Citation
Warren, JJ, Pohlhaus, TJ, Changela, A, Iyer, RR, Modrich, PL, and Beese, LS. "Structure of the human MutSalpha DNA lesion recognition complex." Mol Cell 26.4 (May 25, 2007): 579-592.
PMID
17531815
Source
pubmed
Published In
Molecular Cell
Volume
26
Issue
4
Publish Date
2007
Start Page
579
End Page
592
DOI
10.1016/j.molcel.2007.04.018

Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falciparum. Previously, we have shown that parasites resistant to a tetrahydroquinoline (THQ)-based PFT inhibitor BMS-388891 have mutations leading to amino acid substitutions in PFT that map to the peptide substrate binding domain. We now report the selection of parasites resistant to another THQ PFT inhibitor BMS-339941. In whole cell assays sensitivity to BMS-339941 was reduced by 33-fold in a resistant clone, and biochemical analysis demonstrated a corresponding 33-fold increase in the BMS-339941 K(i) for the mutant PFT enzyme. More detailed kinetic analysis revealed that the mutant enzyme required higher concentration of peptide and farnesyl pyrophosphate substrates for optimum catalysis. Unlike previously characterized parasites resistant to BMS-388891, the resistant parasites have a mutation which is predicted to be in a distinct location of the enzymatic pocket, near the farnesyl pyrophosphate binding pocket. This is the first description of a mutation from any species affecting the farnesyl pyrophosphate binding pocket with reduced efficacy of PFT inhibitors. These data provide further support that PFT is the target of THQ inhibitors in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents to minimize the development of resistant parasites.

Authors
Eastman, RT; White, J; Hucke, O; Yokoyama, K; Verlinde, CLMJ; Hast, MA; Beese, LS; Gelb, MH; Rathod, PK; Van Voorhis, WC
MLA Citation
Eastman, RT, White, J, Hucke, O, Yokoyama, K, Verlinde, CLMJ, Hast, MA, Beese, LS, Gelb, MH, Rathod, PK, and Van Voorhis, WC. "Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase." Mol Biochem Parasitol 152.1 (March 2007): 66-71.
PMID
17208314
Source
pubmed
Published In
Molecular and Biochemical Parasitology
Volume
152
Issue
1
Publish Date
2007
Start Page
66
End Page
71
DOI
10.1016/j.molbiopara.2006.11.012

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase.

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.

Authors
Xu, P; Oum, L; Beese, LS; Geacintov, NE; Broyde, S
MLA Citation
Xu, P, Oum, L, Beese, LS, Geacintov, NE, and Broyde, S. "Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase." Nucleic Acids Res 35.13 (2007): 4275-4288.
PMID
17576677
Source
pubmed
Published In
Nucleic Acids Research
Volume
35
Issue
13
Publish Date
2007
Start Page
4275
End Page
4288
DOI
10.1093/nar/gkm416

The structural basis for the mutagenicity of O(6)-methyl-guanine lesions.

Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine O(6) position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O(6)-methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleotides with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C.O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T.O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.

Authors
Warren, JJ; Forsberg, LJ; Beese, LS
MLA Citation
Warren, JJ, Forsberg, LJ, and Beese, LS. "The structural basis for the mutagenicity of O(6)-methyl-guanine lesions." Proc Natl Acad Sci U S A 103.52 (December 26, 2006): 19701-19706.
PMID
17179038
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
52
Publish Date
2006
Start Page
19701
End Page
19706
DOI
10.1073/pnas.0609580103

The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites.

Periplasmic binding proteins (PBPs) comprise a protein superfamily that is involved in prokaryotic solute transport and chemotaxis. These proteins have been used to engineer reagentless biosensors to detect natural or non-natural ligands. There is considerable interest in obtaining very stable members of this superfamily from thermophilic bacteria to use as robust engineerable parts in biosensor development. Analysis of the recently determined genome sequence of Thermus thermophilus revealed the presence of more than 30 putative PBPs in this thermophile. One of these is annotated as a glucose binding protein (GBP) based on its genetic linkage to genes that are homologous to an ATP-binding cassette glucose transport system, although the PBP sequence is homologous to periplasmic maltose binding proteins (MBPs). Here we present the cloning, over-expression, characterization of cognate ligands, and determination of the X-ray crystal structure of this gene product. We find that it is a very stable (apo-protein Tm value is 100(+/- 2) degrees C; complexes 106(+/- 3) degrees C and 111(+/- 1) degrees C for glucose and galactose, respectively) glucose (Kd value is 0.08(+/- 0.03) microM) and galactose (Kd value is 0.94(+/- 0.04) microM) binding protein. Determination of the X-ray crystal structure revealed that this T. thermophilus glucose binding protein (ttGBP) is structurally homologous to MBPs rather than other GBPs. The di or tri-saccharide ligands in MBPs are accommodated in long relatively shallow grooves. In the ttGBP binding site, this groove is partially filled by two loops and an alpha-helix, which create a buried binding site that allows binding of only monosaccharides. Comparison of ttGBP and MBP provides a clear example of structural adaptations by which the size of ligand binding sites can be controlled in the PBP super family.

Authors
Cuneo, MJ; Changela, A; Warren, JJ; Beese, LS; Hellinga, HW
MLA Citation
Cuneo, MJ, Changela, A, Warren, JJ, Beese, LS, and Hellinga, HW. "The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites." J Mol Biol 362.2 (September 15, 2006): 259-270.
PMID
16904687
Source
pubmed
Published In
Journal of Molecular Biology
Volume
362
Issue
2
Publish Date
2006
Start Page
259
End Page
270
DOI
10.1016/j.jmb.2006.06.084

Conversion of protein farnesyltransferase to a geranylgeranyltransferase.

Posttranslational modifications are essential for the proper function of a number of proteins in the cell. One such modification, the covalent attachment of a single isoprenoid lipid (prenylation), is carried out by the CaaX prenyltransferases, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type-I (GGTase-I). Substrate proteins of these two enzymes are involved in a variety of cellular functions but are largely associated with signal transduction. These modified proteins include members of the Ras superfamily, heterotrimeric G-proteins, centromeric proteins, and a number of proteins involved in nuclear integrity. Although FTase and GGTase-I are highly homologous, they are quite selective for their substrates, particularly for their isoprenoid diphosphate substrates, FPP and GGPP, respectively. Here, we present both crystallographic and kinetic analyses of mutants designed to explore this isoprenoid specificity and demonstrate that this specificity is dependent upon two enzyme residues in the beta subunits of the enzymes, W102beta and Y365beta in FTase (T49beta and F324beta, respectively, in GGTase-I).

Authors
Terry, KL; Casey, PJ; Beese, LS
MLA Citation
Terry, KL, Casey, PJ, and Beese, LS. "Conversion of protein farnesyltransferase to a geranylgeranyltransferase." Biochemistry 45.32 (August 15, 2006): 9746-9755.
PMID
16893176
Source
pubmed
Published In
Biochemistry
Volume
45
Issue
32
Publish Date
2006
Start Page
9746
End Page
9755
DOI
10.1021/bi060295e

Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I.

More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and the gamma subunit of many small heterotrimeric G proteins. This modification is catalyzed by the protein prenyltransferases: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I), and GGTase-II (or RabGGTase). In this review, we examine the structural biology of FTase and GGTase-I (the CaaX prenyltransferases) to establish a framework for understanding the molecular basis of substrate specificity and mechanism. These enzymes have been identified in a number of species, including mammals, fungi, plants, and protists. Prenyltransferase structures include complexes that represent the major steps along the reaction path, as well as a number of complexes with clinically relevant inhibitors. Such complexes may assist in the design of inhibitors that could lead to treatments for cancer, viral infection, and a number of deadly parasitic diseases.

Authors
Lane, KT; Beese, LS
MLA Citation
Lane, KT, and Beese, LS. "Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I." J Lipid Res 47.4 (April 2006): 681-699. (Review)
PMID
16477080
Source
pubmed
Published In
Journal of lipid research
Volume
47
Issue
4
Publish Date
2006
Start Page
681
End Page
699
DOI
10.1194/jlr.R600002-JLR200

Structure of a high fidelity DNA polymerase bound to a benzo[a]pyrene adduct that blocks replication.

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Authors
Hsu, GW; Huang, X; Luneva, NP; Geacintov, NE; Beese, LS
MLA Citation
Hsu, GW, Huang, X, Luneva, NP, Geacintov, NE, and Beese, LS. "Structure of a high fidelity DNA polymerase bound to a benzo[a]pyrene adduct that blocks replication." J Biol Chem 280.5 (February 4, 2005): 3764-3770.
PMID
15548515
Source
pubmed
Published In
The Journal of biological chemistry
Volume
280
Issue
5
Publish Date
2005
Start Page
3764
End Page
3770
DOI
10.1074/jbc.M411276200

Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase.

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.

Authors
Hsu, GW; Kiefer, JR; Burnouf, D; Becherel, OJ; Fuchs, RPP; Beese, LS
MLA Citation
Hsu, GW, Kiefer, JR, Burnouf, D, Becherel, OJ, Fuchs, RPP, and Beese, LS. "Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase." J Biol Chem 279.48 (November 26, 2004): 50280-50285.
PMID
15385534
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
48
Publish Date
2004
Start Page
50280
End Page
50285
DOI
10.1074/jbc.M409224200

Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity.

Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.

Authors
Reid, TS; Terry, KL; Casey, PJ; Beese, LS
MLA Citation
Reid, TS, Terry, KL, Casey, PJ, and Beese, LS. "Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity." J Mol Biol 343.2 (October 15, 2004): 417-433.
PMID
15451670
Source
pubmed
Published In
Journal of Molecular Biology
Volume
343
Issue
2
Publish Date
2004
Start Page
417
End Page
433
DOI
10.1016/j.jmb.2004.08.056

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase.

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.

Authors
Hsu, GW; Ober, M; Carell, T; Beese, LS
MLA Citation
Hsu, GW, Ober, M, Carell, T, and Beese, LS. "Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase." Nature 431.7005 (September 9, 2004): 217-221.
PMID
15322558
Source
pubmed
Published In
Nature
Volume
431
Issue
7005
Publish Date
2004
Start Page
217
End Page
221
DOI
10.1038/nature02908

Crystallographic analysis reveals that anticancer clinical candidate L-778,123 inhibits protein farnesyltransferase and geranylgeranyltransferase-I by different binding modes.

Many signal transduction proteins that control growth, differentiation, and transformation, including Ras GTPase family members, require the covalent attachment of a lipid group by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type-I (GGTase-I) for proper function and for the transforming activity of oncogenic mutants. FTase inhibitors are a new class of potential cancer therapeutics under evaluation in human clinical trials. Here, we present crystal structures of the clinical candidate L-778,123 complexed with mammalian FTase and complexed with the related GGTase-I enzyme. Although FTase and GGTase-I have very similar active sites, L-778,123 adopts different binding modes in the two enzymes; in FTase, L-778,123 is competitive with the protein substrate, whereas in GGTase-I, L-778,123 is competitive with the lipid substrate and inhibitor binding is synergized by tetrahedral anions. A comparison of these complexes reveals that small differences in protein structure can dramatically affect inhibitor binding and selectivity. These structures should facilitate the design of more specific inhibitors toward FTase or GGTase-I. Finally, the binding of a drug and anion together could be applicable for developing new classes of inhibitors.

Authors
Reid, TS; Long, SB; Beese, LS
MLA Citation
Reid, TS, Long, SB, and Beese, LS. "Crystallographic analysis reveals that anticancer clinical candidate L-778,123 inhibits protein farnesyltransferase and geranylgeranyltransferase-I by different binding modes." Biochemistry 43.28 (July 20, 2004): 9000-9008.
PMID
15248757
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
28
Publish Date
2004
Start Page
9000
End Page
9008
DOI
10.1021/bi049280b

Crystal structures of the anticancer clinical candidates R115777 (Tipifarnib) and BMS-214662 complexed with protein farnesyltransferase suggest a mechanism of FTI selectivity.

The search for new cancer therapeutics has identified protein farnesyltransferase (FTase) as a promising drug target. This enzyme attaches isoprenoid lipids to signal transduction proteins involved in growth and differentiation. The two FTase inhibitors (FTIs), R115777 (tipifarnib/Zarnestra) and BMS-214662, have undergone evaluation as cancer therapeutics in phase I and II clinical trials. R115777 has been evaluated in phase III clinical trials and shows indications for the treatment of blood and breast malignancies. Here we present crystal structures of R115777 and BMS-214662 complexed with mammalian FTase. These structures illustrate the molecular mechanism of inhibition and selectivity toward FTase over the related enzyme, protein geranylgeranyltransferase type I (GGTase-I). These results, combined with previous biochemical and structural analyses, identify features of FTase that could be exploited to modulate inhibitor potency and specificity and should aid in the continued development of FTIs as therapeutics for the treatment of cancer and parasitic infections.

Authors
Reid, TS; Beese, LS
MLA Citation
Reid, TS, and Beese, LS. "Crystal structures of the anticancer clinical candidates R115777 (Tipifarnib) and BMS-214662 complexed with protein farnesyltransferase suggest a mechanism of FTI selectivity." Biochemistry 43.22 (June 8, 2004): 6877-6884.
PMID
15170324
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
22
Publish Date
2004
Start Page
6877
End Page
6884
DOI
10.1021/bi049723b

Structures of mismatch replication errors observed in a DNA polymerase.

Accurate DNA replication is essential for genomic stability. One mechanism by which high-fidelity DNA polymerases maintain replication accuracy involves stalling of the polymerase in response to covalent incorporation of mismatched base pairs, thereby favoring subsequent mismatch excision. Some polymerases retain a "short-term memory" of replication errors, responding to mismatches up to four base pairs in from the primer terminus. Here we a present a structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase. Our observations suggest four mechanisms that lead to mismatch-induced stalling of the polymerase. Furthermore, we have observed the effects of extending a mismatch up to six base pairs from the primer terminus and find that long-range distortions in the DNA transmit the presence of the mismatch back to the enzyme active site, suggesting the structural basis for the short-term memory of replication errors.

Authors
Johnson, SJ; Beese, LS
MLA Citation
Johnson, SJ, and Beese, LS. "Structures of mismatch replication errors observed in a DNA polymerase." Cell 116.6 (March 19, 2004): 803-816.
PMID
15035983
Source
pubmed
Published In
Cell
Volume
116
Issue
6
Publish Date
2004
Start Page
803
End Page
816

Structure of mammalian protein geranylgeranyltransferase type-I.

Protein geranylgeranyltransferase type-I (GGTase-I), one of two CaaX prenyltransferases, is an essential enzyme in eukaryotes. GGTase-I catalyzes C-terminal lipidation of >100 proteins, including many GTP- binding regulatory proteins. We present the first structural information for mammalian GGTase-I, including a series of substrate and product complexes that delineate the path of the chemical reaction. These structures reveal that all protein prenyltransferases share a common reaction mechanism and identify specific residues that play a dominant role in determining prenyl group specificity. This hypothesis was confirmed by converting farnesyltransferase (15-C prenyl substrate) into GGTase-I (20-C prenyl substrate) with a single point mutation. GGTase-I discriminates against farnesyl diphosphate (FPP) at the product turnover step through the inability of a 15-C FPP to displace the 20-C prenyl-peptide product. Understanding these key features of specificity is expected to contribute to optimization of anti-cancer and anti-parasite drugs.

Authors
Taylor, JS; Reid, TS; Terry, KL; Casey, PJ; Beese, LS
MLA Citation
Taylor, JS, Reid, TS, Terry, KL, Casey, PJ, and Beese, LS. "Structure of mammalian protein geranylgeranyltransferase type-I." EMBO J 22.22 (November 17, 2003): 5963-5974.
PMID
14609943
Source
pubmed
Published In
EMBO Journal
Volume
22
Issue
22
Publish Date
2003
Start Page
5963
End Page
5974
DOI
10.1093/emboj/cdg571

Dual protein farnesyltransferase-geranylgeranyltransferase-I inhibitors as potential cancer chemotherapeutic agents.

A series of novel diaryl ether lactams have been identified as very potent dual inhibitors of protein farnesyltransferase (FTase) and protein geranylgeranyltransferase I (GGTase-I), enzymes involved in the prenylation of Ras. The structure of the complex formed between one of these compounds and FTase has been determined by X-ray crystallography. These compounds are the first reported to inhibit the prenylation of the important oncogene Ki-Ras4B in vivo. Unfortunately, doses sufficient to achieve this endpoint were rapidly lethal.

Authors
deSolms, SJ; Ciccarone, TM; MacTough, SC; Shaw, AW; Buser, CA; Ellis-Hutchings, M; Fernandes, C; Hamilton, KA; Huber, HE; Kohl, NE; Lobell, RB; Robinson, RG; Tsou, NN; Walsh, ES; Graham, SL; Beese, LS; Taylor, JS
MLA Citation
deSolms, SJ, Ciccarone, TM, MacTough, SC, Shaw, AW, Buser, CA, Ellis-Hutchings, M, Fernandes, C, Hamilton, KA, Huber, HE, Kohl, NE, Lobell, RB, Robinson, RG, Tsou, NN, Walsh, ES, Graham, SL, Beese, LS, and Taylor, JS. "Dual protein farnesyltransferase-geranylgeranyltransferase-I inhibitors as potential cancer chemotherapeutic agents." J Med Chem 46.14 (July 3, 2003): 2973-2984.
PMID
12825937
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
46
Issue
14
Publish Date
2003
Start Page
2973
End Page
2984
DOI
10.1021/jm020587n

Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations.

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.

Authors
Johnson, SJ; Taylor, JS; Beese, LS
MLA Citation
Johnson, SJ, Taylor, JS, and Beese, LS. "Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations." Proc Natl Acad Sci U S A 100.7 (April 1, 2003): 3895-3900.
PMID
12649320
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
100
Issue
7
Publish Date
2003
Start Page
3895
End Page
3900
DOI
10.1073/pnas.0630532100

Reaction path of protein farnesyltransferase at atomic resolution.

Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. FTase inhibitors are currently in clinical trials for the treatment of cancer. Here we present a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. A structural model for the transition state consistent with previous mechanistic studies was also constructed. The processive nature of the reaction suggests the structural basis for the successive addition of two prenyl groups to Rab proteins by the homologous enzyme geranylgeranyltransferase type-II. Finally, known FTase inhibitors seem to differ in their mechanism of inhibiting the enzyme.

Authors
Long, SB; Casey, PJ; Beese, LS
MLA Citation
Long, SB, Casey, PJ, and Beese, LS. "Reaction path of protein farnesyltransferase at atomic resolution." Nature 419.6907 (October 10, 2002): 645-650.
PMID
12374986
Source
pubmed
Published In
Nature
Volume
419
Issue
6907
Publish Date
2002
Start Page
645
End Page
650
DOI
10.1038/nature00986

3-Aminopyrrolidinone farnesyltransferase inhibitors: design of macrocyclic compounds with improved pharmacokinetics and excellent cell potency.

A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date.

Authors
Bell, IM; Gallicchio, SN; Abrams, M; Beese, LS; Beshore, DC; Bhimnathwala, H; Bogusky, MJ; Buser, CA; Culberson, JC; Davide, J; Ellis-Hutchings, M; Fernandes, C; Gibbs, JB; Graham, SL; Hamilton, KA; Hartman, GD; Heimbrook, DC; Homnick, CF; Huber, HE; Huff, JR; Kassahun, K; Koblan, KS; Kohl, NE; Lobell, RB; Lynch, JJ; Robinson, R; Rodrigues, AD; Taylor, JS; Walsh, ES; Williams, TM; Zartman, CB
MLA Citation
Bell, IM, Gallicchio, SN, Abrams, M, Beese, LS, Beshore, DC, Bhimnathwala, H, Bogusky, MJ, Buser, CA, Culberson, JC, Davide, J, Ellis-Hutchings, M, Fernandes, C, Gibbs, JB, Graham, SL, Hamilton, KA, Hartman, GD, Heimbrook, DC, Homnick, CF, Huber, HE, Huff, JR, Kassahun, K, Koblan, KS, Kohl, NE, Lobell, RB, Lynch, JJ, Robinson, R, Rodrigues, AD, Taylor, JS, Walsh, ES, Williams, TM, and Zartman, CB. "3-Aminopyrrolidinone farnesyltransferase inhibitors: design of macrocyclic compounds with improved pharmacokinetics and excellent cell potency." J Med Chem 45.12 (June 6, 2002): 2388-2409.
PMID
12036349
Source
pubmed
Published In
Journal of Medicinal Chemistry
Volume
45
Issue
12
Publish Date
2002
Start Page
2388
End Page
2409

The crystal structure of human protein farnesyltransferase reveals the basis for inhibition by CaaX tetrapeptides and their mimetics.

Protein farnesyltransferase (FTase) catalyzes the attachment of a farnesyl lipid group to the cysteine residue located in the C-terminal tetrapeptide of many essential signal transduction proteins, including members of the Ras superfamily. Farnesylation is essential both for normal functioning of these proteins, and for the transforming activity of oncogenic mutants. Consequently FTase is an important target for anti-cancer therapeutics. Several FTase inhibitors are currently undergoing clinical trials for cancer treatment. Here, we present the crystal structure of human FTase, as well as ternary complexes with the TKCVFM hexapeptide substrate, CVFM non-substrate tetrapeptide, and L-739,750 peptidomimetic with either farnesyl diphosphate (FPP), or a nonreactive analogue. These structures reveal the structural mechanism of FTase inhibition. Some CaaX tetrapeptide inhibitors are not farnesylated, and are more effective inhibitors than farnesylated CaaX tetrapeptides. CVFM and L-739,750 are not farnesylated, because these inhibitors bind in a conformation that is distinct from the TKCVFM hexapeptide substrate. This non-substrate binding mode is stabilized by an ion pair between the peptide N terminus and the alpha-phosphate of the FPP substrate. Conformational mapping calculations reveal the basis for the sequence specificity in the third position of the CaaX motif that determines whether a tetrapeptide is a substrate or non-substrate. The presence of beta-branched amino acids in this position prevents formation of the non-substrate conformation; all other aliphatic amino acids in this position are predicted to form the non-substrate conformation, provided their N terminus is available to bind to the FPP alpha-phosphate. These results may facilitate further development of FTase inhibitors.

Authors
Long, SB; Hancock, PJ; Kral, AM; Hellinga, HW; Beese, LS
MLA Citation
Long, SB, Hancock, PJ, Kral, AM, Hellinga, HW, and Beese, LS. "The crystal structure of human protein farnesyltransferase reveals the basis for inhibition by CaaX tetrapeptides and their mimetics." Proc Natl Acad Sci U S A 98.23 (November 6, 2001): 12948-12953.
PMID
11687658
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
23
Publish Date
2001
Start Page
12948
End Page
12953
DOI
10.1073/pnas.241407898

2 Structure of protein farnesyltransferase

Authors
Terry, KL; Long, SB; Beese, LS
MLA Citation
Terry, KL, Long, SB, and Beese, LS. "2 Structure of protein farnesyltransferase." Enzymes 21.C (2001): 19-46.
Source
scival
Published In
Enzymes
Volume
21
Issue
C
Publish Date
2001
Start Page
19
End Page
46
DOI
10.1016/S1874-6047(01)80015-9

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition.

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.

Authors
Spence, RA; Hightower, KE; Terry, KL; Beese, LS; Fierke, CA; Casey, PJ
MLA Citation
Spence, RA, Hightower, KE, Terry, KL, Beese, LS, Fierke, CA, and Casey, PJ. "Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition." Biochemistry 39.45 (November 14, 2000): 13651-13659.
PMID
11076503
Source
pubmed
Published In
Biochemistry
Volume
39
Issue
45
Publish Date
2000
Start Page
13651
End Page
13659

Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7.

The 2.25 A resolution crystal structure of a pol alpha family (family B) DNA polymerase from the hyperthermophilic marine archaeon Thermococcus sp. 9 degrees N-7 (9 degrees N-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral DNA polymerases. The structure is folded into NH(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha family structures (bacteriophage RB69 and the recently determined Thermococcus gorgonarius), but differ in their relative orientation and conformation. The 9 degrees N-7 polymerase domain structure is reminiscent of the "closed" conformation characteristic of ternary complexes of the pol I polymerase family obtained in the presence of their dNTP and DNA substrates. In the apo-9 degrees N-7 structure, this conformation appears to be stabilized by an ion pair. Thus far, the other apo-pol alpha structures that have been determined adopt open conformations. These results therefore suggest that the pol alpha polymerases undergo a series of conformational transitions during the catalytic cycle similar to those proposed for the pol I family. Furthermore, comparison of the orientations of the fingers and exonuclease (sub)domains relative to the palm subdomain that contains the pol active site suggests that the exonuclease domain and the fingers subdomain of the polymerase can move as a unit and may do so as part of the catalytic cycle. This provides a possible structural explanation for the interdependence of polymerization and editing exonuclease activities unique to pol alpha family polymerases. We suggest that the NH(2)-terminal domain of 9 degrees N-7 pol may be structurally related to an RNA-binding motif, which appears to be conserved among archaeal polymerases. The presence of such a putative RNA- binding domain suggests a mechanism for the observed autoregulation of bacteriophage T4 DNA polymerase synthesis by binding to its own mRNA. Furthermore, conservation of this domain could indicate that such regulation of pol expression may be a characteristic of archaea. Comparion of the 9 degrees N-7 pol structure to its mesostable homolog from bacteriophage RB69 suggests that thermostability is achieved by shortening loops, forming two disulfide bridges, and increasing electrostatic interactions at subdomain interfaces.

Authors
Rodriguez, AC; Park, HW; Mao, C; Beese, LS
MLA Citation
Rodriguez, AC, Park, HW, Mao, C, and Beese, LS. "Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7." J Mol Biol 299.2 (June 2, 2000): 447-462.
PMID
10860752
Source
pubmed
Published In
Journal of Molecular Biology
Volume
299
Issue
2
Publish Date
2000
Start Page
447
End Page
462
DOI
10.1006/jmbi.2000.3728

The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures.

BACKGROUND: The protein farnesyltransferase (FTase) catalyzes addition of the hydrophobic farnesyl isoprenoid to a cysteine residue fourth from the C terminus of several protein acceptors that are essential for cellular signal transduction such as Ras and Rho. This addition is necessary for the biological function of the modified proteins. The majority of Ras-related human cancers are associated with oncogenic variants of K-RasB, which is the highest affinity natural substrate of FTase. Inhibition of FTase causes regression of Ras-mediated tumors in animal models. RESULTS: We present four ternary complexes of rat FTase co-crystallized with farnesyl diphosphate analogs and K-Ras4B peptide substrates. The Ca(1)a(2)X portion of the peptide substrate binds in an extended conformation in the hydrophobic cavity of FTase and coordinates the active site zinc ion. These complexes offer the first view of the polybasic region of the K-Ras4B peptide substrate, which confers the major enhancement of affinity of this substrate. The polybasic region forms a type I beta turn and binds along the rim of the hydrophobic cavity. Removal of the catalytically essential zinc ion results in a dramatically different peptide conformation in which the Ca(1)a(2)X motif adopts a beta turn. A manganese ion binds to the diphosphate mimic of the farnesyl diphosphate analog. CONCLUSIONS: These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.

Authors
Long, SB; Casey, PJ; Beese, LS
MLA Citation
Long, SB, Casey, PJ, and Beese, LS. "The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures." Structure 8.2 (February 15, 2000): 209-222.
PMID
10673434
Source
pubmed
Published In
Structure
Volume
8
Issue
2
Publish Date
2000
Start Page
209
End Page
222

Postpartum hemorrhage: a plan of attack.

Authors
Beese, L
MLA Citation
Beese, L. "Postpartum hemorrhage: a plan of attack." Nursing spectrum (D.C./Baltimore metro ed.) 9.12 (1999): 27--.
PMID
10562241
Source
scival
Published In
Nursing spectrum (D.C./Baltimore metro ed.)
Volume
9
Issue
12
Publish Date
1999
Start Page
27-

Crystal structure of farnesyl protein transferase complexed with a CaaX peptide and farnesyl diphosphate analogue.

The crystallographic structure of acetyl-Cys-Val-Ile-selenoMet-COOH and alpha-hydroxyfarnesylphosphonic acid (alphaHFP) complexed with rat farnesyl protein transferase (FPT) (space group P61, a = b = 174. 13 A, c = 69.71 A, alpha = beta = 90 degrees, gamma = 120 degrees, Rfactor = 21.8%, Rfree = 29.2%, 2.5 A resolution) is reported. In the ternary complex, the bound substrates are within van der Waals contact of each other and the FPT enzyme. alphaHFP binds in an extended conformation in the active-site cavity where positively charged side chains and solvent molecules interact with the phosphate moiety and aromatic side chains pack adjacent to the isoprenoid chain. The backbone of the bound CaaX peptide adopts an extended conformation, and the side chains interact with both FPT and alphaHFP. The cysteine sulfur of the bound peptide coordinates the active-site zinc. Overall, peptide binding and recognition appear to be dominated by side-chain interactions. Comparison of the structures of the ternary complex and unliganded FPT [Park, H., Boduluri, S., Moomaw, J., Casey, P., and Beese, L. (1997) Science 275, 1800-1804] shows that major rearrangements of several active site side chains occur upon substrate binding.

Authors
Strickland, CL; Windsor, WT; Syto, R; Wang, L; Bond, R; Wu, Z; Schwartz, J; Le, HV; Beese, LS; Weber, PC
MLA Citation
Strickland, CL, Windsor, WT, Syto, R, Wang, L, Bond, R, Wu, Z, Schwartz, J, Le, HV, Beese, LS, and Weber, PC. "Crystal structure of farnesyl protein transferase complexed with a CaaX peptide and farnesyl diphosphate analogue." Biochemistry 37.47 (November 24, 1998): 16601-16611.
PMID
9843427
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
47
Publish Date
1998
Start Page
16601
End Page
16611
DOI
10.1021/bi981197z

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon.

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 A with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 A resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Authors
Zhou, M; Mao, C; Rodriguez, AC; Kiefer, JR; Kucera, RB; Beese, LS
MLA Citation
Zhou, M, Mao, C, Rodriguez, AC, Kiefer, JR, Kucera, RB, and Beese, LS. "Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon." Acta Crystallogr D Biol Crystallogr 54.Pt 5 (September 1, 1998): 994-995.
PMID
9757117
Source
pubmed
Published In
Acta Crystallographica Section D
Volume
54
Issue
Pt 5
Publish Date
1998
Start Page
994
End Page
995

Cocrystal structure of protein farnesyltransferase complexed with a farnesyl diphosphate substrate.

Protein farnesyltransferase (FTase) catalyzes the transfer of the hydrophobic farnesyl group from farnesyl diphosphate (FPP) to cellular proteins such as Ras at a cysteine residue near their carboxy-terminus. This process is necessary for the subcellular localization of these proteins to the plasma membrane and is required for the transforming activity of oncogenic variants of Ras, making FTase a prime target for anticancer therapeutics. The high-resolution crystal structure of rat FTase was recently determined, and we present here the X-ray crystal structure of the first complex of FTase with a FPP substrate bound at the active site. The isoprenoid moiety of FPP binds in an extended conformation in a hydrophobic cavity of the beta subunit of the FTase enzyme, and the diphosphate moiety binds to a positively charged cleft at the top of this cavity near the subunit interface. The observed location of the FPP molecule is consistent with mutagenesis data. This binary complex of FTase with FPP leads us to suggest a "molecular ruler" hypothesis for isoprenoid substrate specificity, where the depth of the hydrophobic binding cavity acts as a ruler discriminating between isoprenoids of differing lengths. Although other length isoprenoids may bind in the cavity, only the 15-carbon farnesyl moiety binds with its C1 atom in register with a catalytic zinc ion as required for efficient transfer to the Ras substrate.

Authors
Long, SB; Casey, PJ; Beese, LS
MLA Citation
Long, SB, Casey, PJ, and Beese, LS. "Cocrystal structure of protein farnesyltransferase complexed with a farnesyl diphosphate substrate." Biochemistry 37.27 (July 7, 1998): 9612-9618.
PMID
9657673
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
27
Publish Date
1998
Start Page
9612
End Page
9618
DOI
10.1021/bi980708e

Kinetic analysis of zinc ligand mutants of mammalian protein farnesyltransferase.

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the prenylation of several proteins that are important in cellular regulatory events. A specific residue of FTase, Cys299 in the beta subunit previously identified as essential for zinc binding and catalysis, had been tentatively assigned as one of the zinc ligands. This assignment was subsequently confirmed in the X-ray structure of FTase, which also identified two additional residues, Asp297 and His362 in the beta subunit, as the remaining protein-derived metal ligands. To more fully explore the role of zinc in the catalytic mechanism of FTase, site-directed mutagenesis was performed on these two zinc ligands. Although the abilities of all the mutants to bind the farnesyl diphosphate substrate were similar to that of the wild-type enzyme, all the mutants displayed markedly reduced enzymatic activities and zinc affinities. Steady-state and pre-steady-state kinetic analyses of the residual activities indicated that the rate-limiting step changed from product release in the wild-type enzyme to the chemical step of product formation for three of the mutant enzymes. Additionally, single-turnover experiments indicated that the greatest effect of alteration of zinc ligands for all the mutants was on the product formation step, this being reduced 10(3)-10(5)-fold in the mutant forms compared to the wild-type enzyme. These results confirm a critical involvement of the zinc in catalysis by FTase and support a model in which the metal ion is directly involved in the chemical step of the enzymatic reaction.

Authors
Fu, HW; Beese, LS; Casey, PJ
MLA Citation
Fu, HW, Beese, LS, and Casey, PJ. "Kinetic analysis of zinc ligand mutants of mammalian protein farnesyltransferase." Biochemistry 37.13 (March 31, 1998): 4465-4472.
PMID
9521766
Source
pubmed
Published In
Biochemistry
Volume
37
Issue
13
Publish Date
1998
Start Page
4465
End Page
4472
DOI
10.1021/bi972511c

Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.

DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

Authors
Kiefer, JR; Mao, C; Braman, JC; Beese, LS
MLA Citation
Kiefer, JR, Mao, C, Braman, JC, and Beese, LS. "Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal." Nature 391.6664 (January 15, 1998): 304-307.
PMID
9440698
Source
pubmed
Published In
Nature
Volume
391
Issue
6664
Publish Date
1998
Start Page
304
End Page
307
DOI
10.1038/34693

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 Å with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 Å resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase α (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Authors
Zhou, M; Mao, C; Rodriguez, AC; Kiefer, JR; Kucera, RB; Beese, LS
MLA Citation
Zhou, M, Mao, C, Rodriguez, AC, Kiefer, JR, Kucera, RB, and Beese, LS. "Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon." Acta Crystallographica Section D: Biological Crystallography 54.5 (1998): 994-995.
Source
scival
Published In
Acta Crystallographica Section D
Volume
54
Issue
5
Publish Date
1998
Start Page
994
End Page
995
DOI
10.1107/S0907444998001553

Protein farnesyltransferase.

In the past year, the crystal structure of alpha beta heterodimeric protein farnesyltransferase from rat was reported to a resolution of 2.25 A. Farnesyltransferase catalyzes the essential post-translational lipidation of Ras and several other cellular signal transduction proteins. The structure provides a foundation for understanding the specificity and mechanism of protein prenylation and may aid in the design of new anticancer therapeutics.

Authors
Park, HW; Beese, LS
MLA Citation
Park, HW, and Beese, LS. "Protein farnesyltransferase." Curr Opin Struct Biol 7.6 (December 1997): 873-880. (Review)
PMID
9434909
Source
pubmed
Published In
Current Opinion in Structural Biology
Volume
7
Issue
6
Publish Date
1997
Start Page
873
End Page
880

Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution.

Protein farnesyltransferase (FTase) catalyzes the carboxyl-terminal lipidation of Ras and several other cellular signal transduction proteins. The essential nature of this modification for proper function of these proteins has led to the emergence of FTase as a target for the development of new anticancer therapy. Inhibition of this enzyme suppresses the transformed phenotype in cultured cells and causes tumor regression in animal models. The crystal structure of heterodimeric mammalian FTase was determined at 2.25 angstrom resolution. The structure shows a combination of two unusual domains: a crescent-shaped seven-helical hairpin domain and an alpha-alpha barrel domain. The active site is formed by two clefts that intersect at a bound zinc ion. One cleft contains a nine-residue peptide that may mimic the binding of the Ras substrate; the other cleft is lined with highly conserved aromatic residues appropriate for binding the farnesyl isoprenoid with required specificity.

Authors
Park, HW; Boduluri, SR; Moomaw, JF; Casey, PJ; Beese, LS
MLA Citation
Park, HW, Boduluri, SR, Moomaw, JF, Casey, PJ, and Beese, LS. "Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution." Science 275.5307 (March 21, 1997): 1800-1804.
PMID
9065406
Source
pubmed
Published In
Science
Volume
275
Issue
5307
Publish Date
1997
Start Page
1800
End Page
1804

Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution.

BACKGROUND: The study of DNA polymerases in the Pol l family is central to the understanding of DNA replication and repair. DNA polymerases are used in many molecular biology techniques, including PCR, which require a thermostable polymerase. In order to learn about Pol I function and the basis of thermostability, we undertook structural studies of a new thermostable DNA polymerase. RESULTS: A DNA polymerase large, Klenow-like, fragment from a recently identified thermostable strain of Bacillus stearothermophilus (BF) was cloned, sequenced, overexpressed and characterized. Its crystal structure was determined to 2.1 A resolution by the method of multiple isomorphous replacement. CONCLUSIONS: This structure represents the highest resolution view of a Pol I enzyme obtained to date. Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3'-5' proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft.

Authors
Kiefer, JR; Mao, C; Hansen, CJ; Basehore, SL; Hogrefe, HH; Braman, JC; Beese, LS
MLA Citation
Kiefer, JR, Mao, C, Hansen, CJ, Basehore, SL, Hogrefe, HH, Braman, JC, and Beese, LS. "Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution." Structure 5.1 (January 15, 1997): 95-108.
PMID
9016716
Source
pubmed
Published In
Structure
Volume
5
Issue
1
Publish Date
1997
Start Page
95
End Page
108

Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate.

Crystal structures of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli complexed with deoxynucleoside triphosphate (dNTP) or with pyrophosphate (PPi) determined to 3.9-A resolution by X-ray crystallography show these molecules binding within the cleft of the polymerase domain and surrounded by residues previously implicated in dNTP binding. The dNTP binds adjacent to the O-helix [Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., & Steitz, T. A. (1985a) Nature 313, 762-766] with its triphosphate moiety anchored by three positively charged residues, Arg 754, Arg 682, and Lys 758, plus His 734 and Gln 708. The dNTP binding site observed in the crystal is consistent with the results of chemical modification including cross-linking and is also near many of the amino acid residues whose mutation affects catalysis [Polesky, A. H., Steitz, T. A., Grindley, N. D. F., & Joyce, C. M. (1990) J. Biol. Chem. 265, 14579-14591; Polesky, A. H., Dahlberg, M. E., Benkovic, S. J., Grindley, N. D. F., & Joyce, C. M. (1992) J. Biol. Chem. 267, 8417-8428]. However, we conclude that the position of at least the dNMP moiety of dNTP in the binary complex is not likely to be the same as in its catalytically relevant complex with primer-template DNA.

Authors
Beese, LS; Friedman, JM; Steitz, TA
MLA Citation
Beese, LS, Friedman, JM, and Steitz, TA. "Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate." Biochemistry 32.51 (December 28, 1993): 14095-14101.
PMID
8260491
Source
pubmed
Published In
Biochemistry
Volume
32
Issue
51
Publish Date
1993
Start Page
14095
End Page
14101

Structure of DNA polymerase I Klenow fragment bound to duplex DNA.

Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site.

Authors
Beese, LS; Derbyshire, V; Steitz, TA
MLA Citation
Beese, LS, Derbyshire, V, and Steitz, TA. "Structure of DNA polymerase I Klenow fragment bound to duplex DNA." Science 260.5106 (April 16, 1993): 352-355.
PMID
8469987
Source
pubmed
Published In
Science
Volume
260
Issue
5106
Publish Date
1993
Start Page
352
End Page
355

Two DNA polymerases: HIV reverse transcriptase and the Klenow fragment of Escherichia coli DNA polymerase I.

Authors
Steitz, TA; Smerdon, S; Jäger, J; Wang, J; Kohlstaedt, LA; Friedman, JM; Beese, LS; Rice, PA
MLA Citation
Steitz, TA, Smerdon, S, Jäger, J, Wang, J, Kohlstaedt, LA, Friedman, JM, Beese, LS, and Rice, PA. "Two DNA polymerases: HIV reverse transcriptase and the Klenow fragment of Escherichia coli DNA polymerase I." Cold Spring Harb Symp Quant Biol 58 (1993): 495-504.
PMID
7525146
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
58
Publish Date
1993
Start Page
495
End Page
504

Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.

The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5' exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and 3.1 A resolution respectively. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 A from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.

Authors
Beese, LS; Steitz, TA
MLA Citation
Beese, LS, and Steitz, TA. "Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism." EMBO J 10.1 (January 1991): 25-33.
PMID
1989886
Source
pubmed
Published In
EMBO Journal
Volume
10
Issue
1
Publish Date
1991
Start Page
25
End Page
33

Correction for specimen movement after acquisition of element-specific electron microprobe images.

Because a long time is generally required to generate X-ray maps of specific elements by electron beam methods, images are subject to a loss of resolution due to stage movement. Methods have been previously described for correcting stage drift during exposure by sensing the drift and deflecting the beam to follow the stage; but these methods require modifications of the equipment. When the drift is not excessive, it is possible to correct a series of images after the exposure series is finished. Here we demonstrate two methods for correcting the drift, one based on manual assignment of specimen position and one on the use of cross-correlation functions to determine objectively the misalignment of images in the series. The success of the methods is illustrated in calcium-specific images of a bone section that show the collagen periodicity after drift correction.

Authors
Lamvik, MK; Ingram, P; Menon, RG; Beese, LS; Davilla, SD; LeFurgey, A
MLA Citation
Lamvik, MK, Ingram, P, Menon, RG, Beese, LS, Davilla, SD, and LeFurgey, A. "Correction for specimen movement after acquisition of element-specific electron microprobe images." J Microsc 156.Pt 2 (November 1989): 183-190.
PMID
2593148
Source
pubmed
Published In
Journal of Microscopy
Volume
156
Issue
Pt 2
Publish Date
1989
Start Page
183
End Page
190

Correction for specimen movement after acquisition of element-specific electron microprobe images

Authors
Lamvik, MK; Ingram, P; Menon, RG; Beese, LS; Davilla, SD; LeFurgey, A
MLA Citation
Lamvik, MK, Ingram, P, Menon, RG, Beese, LS, Davilla, SD, and LeFurgey, A. "Correction for specimen movement after acquisition of element-specific electron microprobe images." Journal of Microscopy 156.2 (1989): 183-190.
Source
scival
Published In
Journal of Microscopy
Volume
156
Issue
2
Publish Date
1989
Start Page
183
End Page
190

Cocrystal structure of an editing complex of Klenow fragment with DNA.

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.

Authors
Freemont, PS; Friedman, JM; Beese, LS; Sanderson, MR; Steitz, TA
MLA Citation
Freemont, PS, Friedman, JM, Beese, LS, Sanderson, MR, and Steitz, TA. "Cocrystal structure of an editing complex of Klenow fragment with DNA." Proc Natl Acad Sci U S A 85.23 (December 1988): 8924-8928.
PMID
3194400
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
85
Issue
23
Publish Date
1988
Start Page
8924
End Page
8928

Genetic and Crystallographic Studies of the 3′,5′- exonucleolytic site of DNA polymerase I

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3′,5′-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changs at the mutationn sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fagment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonudease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

Authors
Derbyshire, V; Freemont, PS; Sanderson, MR; Beese, L; Friedman, JM; Joyce, CM; Steitz, TA
MLA Citation
Derbyshire, V, Freemont, PS, Sanderson, MR, Beese, L, Friedman, JM, Joyce, CM, and Steitz, TA. "Genetic and Crystallographic Studies of the 3′,5′- exonucleolytic site of DNA polymerase I." Science 240.4849 (1988): 199-201.
PMID
2832946
Source
scival
Published In
Science
Volume
240
Issue
4849
Publish Date
1988
Start Page
199
End Page
201

Structure of microtubules with reduced hydration. Comparison of results from X-ray diffraction and electron microscopy

A recent model for the structure of microtubules is used to interpret X-ray fiber diffraction patterns from microtubules, obtained under various conditions. The results suggest that tubulin may undergo conformational changes under conditions of reduced water-activity. Such changes could account for some of the differences in the structure of tubulin as determined by electron microscopy and X-ray diffraction. © 1987.

Authors
Beese, L; Stubbs, G; Thomas, J; Cohen, C
MLA Citation
Beese, L, Stubbs, G, Thomas, J, and Cohen, C. "Structure of microtubules with reduced hydration. Comparison of results from X-ray diffraction and electron microscopy." Journal of Molecular Biology 196.3 (1987): 575-580.
PMID
3681968
Source
scival
Published In
Journal of Molecular Biology
Volume
196
Issue
3
Publish Date
1987
Start Page
575
End Page
580

Structural studies of Klenow fragment: An enzyme with two active sites

Authors
Steitz, TA; Beese, L; Freemont, PS; Friedman, JM; Sanderson, MR
MLA Citation
Steitz, TA, Beese, L, Freemont, PS, Friedman, JM, and Sanderson, MR. "Structural studies of Klenow fragment: An enzyme with two active sites." Cold Spring Harbor Symposia on Quantitative Biology 52 (1987): 465-471.
PMID
3331343
Source
scival
Published In
Cold Spring Harbor Symposia on Quantitative Biology
Volume
52
Publish Date
1987
Start Page
465
End Page
471

Microtubule structure at 18 Å resolution

A model for the structure of microtubules at a resolution of 18 Å (1 Å = 0.1 nm) is described, based on X-ray fiber diffraction data from hydrated reassembled calf brain microtubules. The model was derived by an iterative solvent flattening refinement procedure, with initial phases based on those determined by electron microscopy. The major microtubule surface grooves are those defining the protofilaments, which form a hollow cylinder of maximum diameter 300 Å. Strong electron density fluctuations in the microtubule wall are interpreted as evidence for a domain structure within the tubulin subunit. The arrangement of domains is such that the tubulin molecule could be quite flexible at the domain connections; thus, slight changes in this arrangement could account for the unusual polymorphism of tubulin assemblies. © 1987.

Authors
Beese, L; Stubbs, G; Cohen, C
MLA Citation
Beese, L, Stubbs, G, and Cohen, C. "Microtubule structure at 18 Å resolution." Journal of Molecular Biology 194.2 (1987): 257-264.
PMID
3612805
Source
scival
Published In
Journal of Molecular Biology
Volume
194
Issue
2
Publish Date
1987
Start Page
257
End Page
264

Contact x-ray microscopy. A new technique for imaging cellular fine structure.

Contact x-ray microscopy potentially allows living, wet cells to be visualized at a resolution of up to 100 A. Furthermore, differential absorption by specific elements permits the study of the distribution of those elements in biological specimens. In contact x-ray microscopy, soft x-rays (10 A to 100 A) pass through a biological sample and expose an underlying x-ray sensitive polymer (resist), producing an image that reflects the photon absorbance within the specimen. The high penetrating power of soft x-ray enables images to be obtained from specimens up to several microns thick. In this paper, the technique is described, some of the areas currently under study are considered, and biological examples of the use of contact x-ray microscopy are given.

Authors
Beese, L; Feder, R; Sayre, D
MLA Citation
Beese, L, Feder, R, and Sayre, D. "Contact x-ray microscopy. A new technique for imaging cellular fine structure." Biophysical journal 49.1 (1986): 259-268.
PMID
3955174
Source
scival
Published In
Biophysical journal
Volume
49
Issue
1
Publish Date
1986
Start Page
259
End Page
268
DOI
10.1016/S0006-3495(86)83639-6

Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin

Scallop thin filaments decorated with proteolytic fragments of scallop myosin display two forms of "arrowhead" complex by electron microscopy, depending on the presence or absence of the regulatory light chain. The arrowhead pattern obtained with heavy meromyosin or myosin subfragment-1 from which this light chain has been removed resembles that previously obtained by Moore et al. (1970) with rabbit myosin subfragment-1. We term this form "blunted". When the regulatory light chain is present, the arrowhead profiles look distinctly different and appear more "barbed". The attachment of individual heads or pairs of heads can also be observed when lower concentrations of myosin fragments are used. The angles of attachment and the shape of the heads can be seen in this case without the superposition that occurs in fully decorated filaments. Both heads of heavy mero-myosin appear to attach to the same actin strand of a single thin filament, usually to adjacent actin monomers. The heads are long (about 20 nm), narrow (about 4 nm wide), and distinctly curved. Both attach at approximately the same angle to actin and join to the rod by apparent elongation and bending of the leading head of the pair. There is some evidence that the binding of myosin heads to thin filaments may be co-operative. In preliminary experiments, we have duplicated most of the above observations using rabbit skeletal muscle proteins. © 1980.

Authors
Craig, R; Szent-Györgyi, AG; Beese, L; Flicker, P; Vibert, P; Cohen, C
MLA Citation
Craig, R, Szent-Györgyi, AG, Beese, L, Flicker, P, Vibert, P, and Cohen, C. "Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin." Journal of Molecular Biology 140.1 (1980): 35-55.
PMID
6997502
Source
scival
Published In
Journal of Molecular Biology
Volume
140
Issue
1
Publish Date
1980
Start Page
35
End Page
55

Carbohydrate metabolism disorders in kidney diseases

Authors
Garmendia, F; Rojas, MI; Valdivia, H; Lorena, B; García, R
MLA Citation
Garmendia, F, Rojas, MI, Valdivia, H, Lorena, B, and García, R. "Carbohydrate metabolism disorders in kidney diseases." Anales del Programa Academico de Medicina 52.3 (1969): 121-130.
Source
scival
Published In
Anales del Programa Academico de Medicina
Volume
52
Issue
3
Publish Date
1969
Start Page
121
End Page
130
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