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Lee, Seok-Yong

Positions:

Associate Professor of Biochemistry

Biochemistry
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

B.S. 1998

B.S. — Yonsei University

Ph.D. 2003

Ph.D. — University of California at Berkeley

News:

Grants:

Structure, function, and pharmacology of neuronal membrane transport proteins

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2016
End Date
August 31, 2024

Structural and Mechanistic Characterization of MraY Catalysis and Inhibition

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2017
End Date
November 30, 2020

Development of novel therapeutics for pain and itch relief

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2014
End Date
July 31, 2018

Structure and Function of Concentrative Nucleoside Transporters

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
May 01, 2013
End Date
July 31, 2017

Pharmacology and biophysics of the voltage-gated sodium channel Nav1.7: a therapeutic target for pain

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 30, 2011
End Date
June 30, 2016

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

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

Automated detection of protein crystals in high-throughput crystallography experiments

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

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

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

NIGMS award, 56th Biophysical Society Annual Meeting. NIGMS.

Type
International
Awarded By
NIGMS
Date
January 01, 2012

Basil O'connor Starter Scholar Research Award. March of Dimes Foundation.

Type
National
Awarded By
March of Dimes Foundation
Date
January 01, 2011

NIH Director’s New Innovator Award. NIH.

Type
National
Awarded By
NIH
Date
January 01, 2011

Sloan Research Fellowship-Neuroscience. Alfred P. Sloan Foundation.

Type
National
Awarded By
Alfred P. Sloan Foundation
Date
January 01, 2011

Klingenstein Fellowship Award in the Neurosciences. Klingenstein Foundation.

Type
National
Awarded By
Klingenstein Foundation
Date
January 01, 2010

Mallinckrodt Scholar Award. Mallinckrodt Foundation.

Type
National
Awarded By
Mallinckrodt Foundation
Date
January 01, 2010

McKnight Scholar Award. McKnight Foundation.

Type
National
Awarded By
McKnight Foundation
Date
January 01, 2010

Publications:

Crystal structure of the MOP flippase MurJ in an inward-facing conformation

Authors
Kuk, ACY; Mashalidis, EH; Lee, S-Y
MLA Citation
Kuk, ACY, Mashalidis, EH, and Lee, S-Y. "Crystal structure of the MOP flippase MurJ in an inward-facing conformation." Nature Structural & Molecular Biology 24.2 (December 26, 2016): 171-176.
Source
crossref
Published In
Nature Structural & Molecular Biology
Volume
24
Issue
2
Publish Date
2016
Start Page
171
End Page
176
DOI
10.1038/nsmb.3346

Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins.

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage-gated Na(+) channels (NaV channels) triggers action potentials, a process that is regulated via the interactions of the channels' intercellular C-termini with auxiliary proteins and/or Ca(2+) . The molecular and structural details for how Ca(2+) and/or auxiliary proteins modulate NaV channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca(2+) acts directly upon the NaV channel or through interacting proteins, such as the Ca(2+) binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of NaV intracellular C-termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca(2+) affects NaV function, and how altered Ca(2+) -dependent or FHF-mediated regulation of NaV channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.

Authors
Pitt, GS; Lee, S-Y
MLA Citation
Pitt, GS, and Lee, S-Y. "Current view on regulation of voltage-gated sodium channels by calcium and auxiliary proteins." Protein science : a publication of the Protein Society 25.9 (September 2016): 1573-1584.
PMID
27262167
Source
epmc
Published In
Protein Science
Volume
25
Issue
9
Publish Date
2016
Start Page
1573
End Page
1584
DOI
10.1002/pro.2960

Structural insights into inhibition of lipid I production in bacterial cell wall synthesis.

Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraYAA) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraYAA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraYAA. Surprisingly, MD2 does not interact with three acidic residues or the Mg(2+) cofactor required for catalysis, suggesting that MD2 binds to MraYAA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraYAA, including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. These findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO.

Authors
Chung, BC; Mashalidis, EH; Tanino, T; Kim, M; Matsuda, A; Hong, J; Ichikawa, S; Lee, S-Y
MLA Citation
Chung, BC, Mashalidis, EH, Tanino, T, Kim, M, Matsuda, A, Hong, J, Ichikawa, S, and Lee, S-Y. "Structural insights into inhibition of lipid I production in bacterial cell wall synthesis." Nature 533.7604 (May 2016): 557-560.
PMID
27088606
Source
epmc
Published In
Nature
Volume
533
Issue
7604
Publish Date
2016
Start Page
557
End Page
560
DOI
10.1038/nature17636

Cryo-electron microscopy structure of the TRPV2 ion channel.

Transient receptor potential vanilloid (TRPV) cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. TRPV2 has been implicated in many biological functions, including somatosensation, osmosensation and innate immunity. Here we present the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at a nominal resolution of ∼4 Å. In the TRPV2 structure, the transmembrane segment 6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6.

Authors
Zubcevic, L; Herzik, MA; Chung, BC; Liu, Z; Lander, GC; Lee, S-Y
MLA Citation
Zubcevic, L, Herzik, MA, Chung, BC, Liu, Z, Lander, GC, and Lee, S-Y. "Cryo-electron microscopy structure of the TRPV2 ion channel." Nature structural & molecular biology 23.2 (February 2016): 180-186.
PMID
26779611
Source
epmc
Published In
Nature Structural & Molecular Biology
Volume
23
Issue
2
Publish Date
2016
Start Page
180
End Page
186
DOI
10.1038/nsmb.3159

Liposome reconstitution and transport assay for recombinant transportersLiposome Reconstitution and Transport Assay for Recombinant Transporters

© 2015 Elsevier Inc. All rights reserved.Secondary active transporters are responsible for the cellular uptake of many biologically important molecules, including neurotransmitters, nutrients, and drugs. Because of their physiological and clinical importance, a method for assessing their transport activity in vitro is necessary to gain a better understanding of how these transporters function at the molecular level. In this chapter, we describe a protocol for reconstituting the concentrative nucleoside transporter from Vibrio cholerae into proteoliposomes. We then describe a radiolabeled substrate uptake assay that can be used to functionally characterize the transporter. These methods are relatively common and can be applied to other secondary active transporters, with or without some modification.

Authors
Johnson, ZL; Lee, SY; Johnson, ZL; Lee, S-Y
MLA Citation
Johnson, ZL, Lee, SY, Johnson, ZL, and Lee, S-Y. "Liposome reconstitution and transport assay for recombinant transportersLiposome Reconstitution and Transport Assay for Recombinant Transporters (PublishedAccepted)." Methods in Enzymology 556 (January 1, 2015): 373-383.
Source
scopus
Published In
Methods in Enzymology
Volume
556
Publish Date
2015
Start Page
373
End Page
383
DOI
10.1016/bs.mie.2014.11.048

Structural analyses of Ca²⁺/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation.

Ca(2+) regulates voltage-gated Na(+) (NaV) channels, and perturbed Ca(2+) regulation of NaV function is associated with epilepsy syndromes, autism and cardiac arrhythmias. Understanding the disease mechanisms, however, has been hindered by a lack of structural information and competing models for how Ca(2+) affects NaV channel function. Here we report the crystal structures of two ternary complexes of a human NaV cytosolic C-terminal domain (CTD), a fibroblast growth factor homologous factor and Ca(2+)/calmodulin (Ca(2+)/CaM). These structures rule out direct binding of Ca(2+) to the NaV CTD and uncover new contacts between CaM and the NaV CTD. Probing these new contacts with biochemical and functional experiments allows us to propose a mechanism by which Ca(2+) could regulate NaV channels. Further, our model provides hints towards understanding the molecular basis of the neurologic disorders and cardiac arrhythmias caused by NaV channel mutations.

Authors
Wang, C; Chung, BC; Yan, H; Wang, H-G; Lee, S-Y; Pitt, GS
MLA Citation
Wang, C, Chung, BC, Yan, H, Wang, H-G, Lee, S-Y, and Pitt, GS. "Structural analyses of Ca²⁺/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation." Nature communications 5 (September 18, 2014): 4896-.
PMID
25232683
Source
epmc
Published In
Nature Communications
Volume
5
Publish Date
2014
Start Page
4896
DOI
10.1038/ncomms5896

Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters.

Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively.

Authors
Johnson, ZL; Lee, J-H; Lee, K; Lee, M; Kwon, D-Y; Hong, J; Lee, S-Y
MLA Citation
Johnson, ZL, Lee, J-H, Lee, K, Lee, M, Kwon, D-Y, Hong, J, and Lee, S-Y. "Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters." eLife 3 (July 31, 2014): e03604-.
PMID
25082345
Source
epmc
Published In
eLife
Volume
3
Publish Date
2014
Start Page
e03604
DOI
10.7554/elife.03604

Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

Authors
Johnson, ZL; Lee, J-H; Lee, K; Lee, M; Kwon, D-Y; Hong, J; Lee, S-Y
MLA Citation
Johnson, ZL, Lee, J-H, Lee, K, Lee, M, Kwon, D-Y, Hong, J, and Lee, S-Y. "Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters." ELIFE 3 (July 31, 2014).
Source
wos-lite
Published In
eLife
Volume
3
Publish Date
2014
DOI
10.7554/eLife.03604

A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief.

Voltage-gated sodium (NaV) channels control the upstroke of the action potentials in excitable cells. Multiple studies have shown distinct roles of NaV channel subtypes in human physiology and diseases, but subtype-specific therapeutics are lacking and the current efforts have been limited to small molecules. Here, we present a monoclonal antibody that targets the voltage-sensor paddle of NaV1.7, the subtype critical for pain sensation. This antibody not only inhibits NaV1.7 with high selectivity, but also effectively suppresses inflammatory and neuropathic pain in mice. Interestingly, the antibody inhibits acute and chronic itch despite well-documented differences in pain and itch modulation. Using this antibody, we discovered that NaV1.7 plays a key role in spinal cord nociceptive and pruriceptive synaptic transmission. Our studies reveal that NaV1.7 is a target for itch management, and the antibody has therapeutic potential for suppressing pain and itch. Our antibody strategy may have broad applications for voltage-gated cation channels.

Authors
Lee, J-H; Park, C-K; Chen, G; Han, Q; Xie, R-G; Liu, T; Ji, R-R; Lee, S-Y
MLA Citation
Lee, J-H, Park, C-K, Chen, G, Han, Q, Xie, R-G, Liu, T, Ji, R-R, and Lee, S-Y. "A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief." Cell 157.6 (June 2014): 1393-1404.
PMID
24856969
Source
epmc
Published In
Cell
Volume
157
Issue
6
Publish Date
2014
Start Page
1393
End Page
1404
DOI
10.1016/j.cell.2014.03.064

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

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

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

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

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

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

Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin.

Voltage-gated Na⁺ (Na(V)) channels initiate neuronal action potentials. Na(V) channels are composed of a transmembrane domain responsible for voltage-dependent Na⁺ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human Na(V) CTD, an FHF, and Ca²⁺-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in Na(V) channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual Na(V) CTD isoforms for distinctive FHFs.

Authors
Wang, C; Chung, BC; Yan, H; Lee, S-Y; Pitt, GS
MLA Citation
Wang, C, Chung, BC, Yan, H, Lee, S-Y, and Pitt, GS. "Crystal structure of the ternary complex of a NaV C-terminal domain, a fibroblast growth factor homologous factor, and calmodulin." Structure 20.7 (July 3, 2012): 1167-1176.
PMID
22705208
Source
pubmed
Published In
Structure
Volume
20
Issue
7
Publish Date
2012
Start Page
1167
End Page
1176
DOI
10.1016/j.str.2012.05.001

Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes. © 2012 Macmillan Publishers Limited. All rights reserved.

Authors
Johnson, ZL; Cheong, C-G; Lee, S-Y
MLA Citation
Johnson, ZL, Cheong, C-G, and Lee, S-Y. "Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å." Nature 483.7390 (2012): 489-493.
PMID
22407322
Source
scival
Published In
Nature
Volume
483
Issue
7390
Publish Date
2012
Start Page
489
End Page
493
DOI
10.1038/nature10882

Functional Reconstitution of Purified Human Hv1 H+ Channels

Voltage-dependent H+ (Hv) channels mediate proton conduction into and out of cells under the control of membrane voltage. Hv channels are unusual compared to voltage-dependent K+, Na+, and Ca2+ channels in that Hv channel genes encode a voltage sensor domain (VSD) without a pore domain. The H+ currents observed when Hv channels are expressed heterologously suggest that the VSD itself provides the pathway for proton conduction. In order to exclude the possibility that the Hv channel VSD assembles with an as yet unknown protein in the cell membrane as a requirement for H+ conduction, we have purified Hv channels to homogeneity and reconstituted them into synthetic lipid liposomes. The Hv channel VSD by itself supports H+ flux. © 2009 Elsevier Ltd. All rights reserved.

Authors
Lee, S-Y; Letts, JA; MacKinnon, R
MLA Citation
Lee, S-Y, Letts, JA, and MacKinnon, R. "Functional Reconstitution of Purified Human Hv1 H+ Channels." Journal of Molecular Biology 387.5 (2009): 1055-1060.
PMID
19233200
Source
scival
Published In
Journal of Molecular Biology
Volume
387
Issue
5
Publish Date
2009
Start Page
1055
End Page
1060
DOI
10.1016/j.jmb.2009.02.034

Two Separate Interfaces between the Voltage Sensor and Pore Are Required for the Function of Voltage-Dependent K + Channels

Voltage-dependent K + (Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate. © 2009 Lee et al.

Authors
Lee, S-Y; Banerjee, A; MacKinnon, R
MLA Citation
Lee, S-Y, Banerjee, A, and MacKinnon, R. "Two Separate Interfaces between the Voltage Sensor and Pore Are Required for the Function of Voltage-Dependent K + Channels." PLoS Biology 7.3 (2009).
PMID
19260762
Source
scival
Published In
PLoS biology
Volume
7
Issue
3
Publish Date
2009
DOI
10.1371/journal.pbio.1000047

Two separate interfaces between the Voltage sensor and pore are required for the function of Voltage-dependent K+ channels

Voltage-dependent K+(Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltagesensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate. Copyright: © 2009 Lee et al. This is an open-access article distributed under the.

Authors
Lee, S-Y; Banerjee, A; Mackinnon, R
MLA Citation
Lee, S-Y, Banerjee, A, and Mackinnon, R. "Two separate interfaces between the Voltage sensor and pore are required for the function of Voltage-dependent K+ channels." PLoS Biology 7.3 (2009): 0676-0686.
Source
scival
Published In
PLoS biology
Volume
7
Issue
3
Publish Date
2009
Start Page
0676
End Page
0686
DOI
10.1371/journal.pbio.1000047-S.pdf

Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1

In voltage-gated Na+, K+, and Ca2+ channels, four voltage-sensor domains operate on a central pore domain in response to membrane voltage. In contrast, the voltage-gated proton channel (Hv) contains only a voltage-sensor domain, lacking a separate pore domain. The subunit stoichiometry and organization of Hv has been unknown. Here, we show that human Hv1 forms a dimer in the membrane and define regions that are close to the dimer interface by using cysteine cross-linking. Two dimeric interfaces appear to exist in Hv1, one mediated by S1 and the adjacent extracellular loop, and the other mediated by a putative intracellular coiled-coil domain. It may be significant that Hv1 uses for its dimer interface a surface that corresponds to the interface between the voltage sensor and pore in Kv channels. © 2008 by The National Academy of Sciences of the USA.

Authors
Lee, S-Y; Letts, JA; MacKinnon, R
MLA Citation
Lee, S-Y, Letts, JA, and MacKinnon, R. "Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1." Proceedings of the National Academy of Sciences of the United States of America 105.22 (2008): 7692-7695.
PMID
18509058
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
105
Issue
22
Publish Date
2008
Start Page
7692
End Page
7695
DOI
10.1073/pnas.0803277105

Structure of the KvAP voltage-dependent K+ channel and its on the lipid membrane

Voltage-dependent ion channels gate open in response to changes in cell membrane voltage. This form of gating permits the propagation of action potentials. We present two structures of the voltage-dependent K+ channel KvAP, in complex with monoclonal Fv fragments (3.9 Å) and without antibody fragments (8 Å). We also studied KvAP with disulfide cross-bridges in lipid membranes. Analyzing these data in the context of the crystal structure of Kv1.2 and EPR data on KvAP we reach the following conclusions: (i) KvAP is similar in structure to Kv1.2 with a very modest difference in the orientation of its voltage sensor; (ii) mAb fragments are not the source of non-native conformations of KvAP in crystal structures; (iii) because KvAP contains separate loosely adherent domains, a lipid membrane is required to maintain their correct relative orientations, and (iv) the model of KvAP is consistent with the proposal of voltage sensing through the movement of an arginine-containing helix-turn-helix element at the protein-lipid interface. © 2005 by The National Academy of Sciences of the USA.

Authors
Lee, S-Y; Lee, A; Chen, J; MacKinnon, R
MLA Citation
Lee, S-Y, Lee, A, Chen, J, and MacKinnon, R. "Structure of the KvAP voltage-dependent K+ channel and its on the lipid membrane." Proceedings of the National Academy of Sciences of the United States of America 102.43 (2005): 15441-15446.
PMID
16223877
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
43
Publish Date
2005
Start Page
15441
End Page
15446
DOI
10.1073/pnas.0507651102

Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues

Dihydrofolate reductase (DHFR) from a moderate thermophilic organism, Bacillus stearothermophilus, has been cloned and expressed. Physical characterization of the protein (BsDHFR) indicates that it is a monomeric protein with a molecular mass of 18 694.6 Da (0.8), coincident with the mass of 18 694.67 Da calculated from the primary sequence. Determination of the X-ray structure of BsDHFR provides the first structure for a monomeric DHFR from a thermophilic organism, indicating a high degree of conservation of structure in relation to all chromosomal DHFRs. Structurally based sequence alignment of DHFRs indicates the following levels of sequence identity and similarity for BsDHFR: 38 and 58% with Escherichia coli, 35 and 56% with Lactobacillus casei, and 23 and 40% with Thermotoga maritima, respectively. Steady state kinetic isotope effect studies indicate an ordered kinetic mechanism at elevated temperatures, with NADPH binding first to the enzyme. This converts to a more random mechanism at reduced temperatures, reflected in a greatly reduced K m for dihydrofolate at 20 °C in relation to that at 60 °C. A reduction in either temperature or pH reduces the degree to which the hydride transfer step is rate-determining for the second-order reaction of DHF with the enzyme-NADPH binary complex. Transient state kinetics have been used to study the temperature dependence of the isotope effect on hydride transfer at pH 9 between 10 and 50 °C. The data support rate-limiting hydride transfer with a moderate enthalpy of activation (Ea = 5.5 kcal/mol) and a somewhat greater temperature dependence for the kinetic isotope effect than predicted from classical behavior [AH/AD = 0.57 (0.15)]. Comparison of kinetic parameters for BsDHFR to published data for DHFR from E. coli and T. maritima shows a decreasing trend in efficiency of hydride transfer with increasing thermophilicity of the protein. These results are discussed in the context of the capacity of each enzyme to optimize H-tunneling from donor (NADPH) to acceptor (DHF) substrates. © 2005 American Chemical Society.

Authors
Kim, HS; Damo, SM; Lee, S-Y; Wemmer, D; Klinman, JP
MLA Citation
Kim, HS, Damo, SM, Lee, S-Y, Wemmer, D, and Klinman, JP. "Structure and hydride transfer mechanism of a moderate thermophilic dihydrofolate reductase from Bacillus stearothermophilus and comparison to its mesophilic and hyperthermophilic homologues." Biochemistry 44.34 (2005): 11428-11439.
PMID
16114879
Source
scival
Published In
Biochemistry
Volume
44
Issue
34
Publish Date
2005
Start Page
11428
End Page
11439
DOI
10.1021/bi050630j

YbiV from Escherichia coli K12 is a HAD phosphatase

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized YbiV as a HAD phosphatase. The crystal structure of YbiV reveals a two-domain protein, one with the characteristic HAD hydrolase fold, the other an inserted α/β fold. In an effort to understand the mechanism, we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3), which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate-binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate YbiV may be a sugar phosphatase, which is supported by biochemical assays that measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate. © 2005 Wiley-Liss, Inc.

Authors
Roberts, A; Lee, S-Y; McCullagh, E; Silversmith, RE; Wemmer, DE
MLA Citation
Roberts, A, Lee, S-Y, McCullagh, E, Silversmith, RE, and Wemmer, DE. "YbiV from Escherichia coli K12 is a HAD phosphatase." Proteins: Structure, Function and Genetics 58.4 (2005): 790-801.
PMID
15657928
Source
scival
Published In
Proteins: Structure, Function and Genetics
Volume
58
Issue
4
Publish Date
2005
Start Page
790
End Page
801
DOI
10.1002/prot.20267

A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom

Venomous animals produce small protein toxins that inhibit ion channels with high affinity. In several well-studied cases the inhibitory proteins are water-soluble and bind at a channel's aqueous-exposed extracellular surface. Here we show that a voltage-sensor toxin (VSTX1) from the Chilean Rose Tarantula (Grammostola spatulata) reaches its target by partitioning into the lipid membrane. Lipid membrane partitioning serves two purposes: to localize the toxin in the membrane where the voltage sensor resides and to exploit the free energy of partitioning to achieve apparent high-affinity inhibition. VSTX1, small hydrophobic poisons and anaesthetic molecules reveal a common theme of voltage sensor inhibition through lipid membrane access. The apparent requirement for such access is consistent with the recent proposal that the sensor in voltage-dependent K+ channels is located at the membrane-protein interface.

Authors
Lee, SY; MacKinnon, R
MLA Citation
Lee, SY, and MacKinnon, R. "A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom." Nature 430.6996 (2004): 232-235.
PMID
15241419
Source
scival
Published In
Nature
Volume
430
Issue
6996
Publish Date
2004
Start Page
232
End Page
235
DOI
10.1038/nature02632

High-resolution solution structure of the beryllofluoride-activated NtrC receiver domain

Bacterial receiver domains mediate the cellular response to environmental changes through conformational changes induced by phosphorylation of a conserved aspartate residue. While the structures of several activated receiver domains have recently been determined, there is substantial variation in the conformational changes occurring upon activation. Here we present the high-resolution structure of the activated NtrC receiver domain (BeF3--NtrCr complex) determined using NMR data, including residual dipolar couplings, yielding a family of structures with a backbone rmsd of 0.57 ± 0.08 Å, which is compared with the previous lower-resolution structure of the phosphorylated protein. Both phosphorylation and beryllofluoride addition induce a shift in register and an axial rotation of α-helix 4. In this high-resolution structure, we are able to observe a concerted change in the positions of Thr82 and Tyr101; this correlated change in two conserved residues (termed Y-T coupling) has been considered a general feature of the conformational change in receiver domains upon activation. In NtrC, this correlated side chain shift, leading to the helix reorientation, is distinctly different from the smaller reorganization seen in other activated receiver domains, and involves numerous other residues which do not participate in conformational changes seen in the other systems. Titration of the activated receiver domain with peptides from the NtrC ATPase domain provides direct evidence for interactions on the rearranged face of the receiver domain, which are likely to be responsible for enabling assembly into the active aggregate. Analysis of the active structure also suggests that His84 may play a role in controlling the phosphate hydrolysis rate.

Authors
Hastings, CA; Lee, S-Y; Cho, HS; Yan, D; Kustu, S; Wemmer, DE
MLA Citation
Hastings, CA, Lee, S-Y, Cho, HS, Yan, D, Kustu, S, and Wemmer, DE. "High-resolution solution structure of the beryllofluoride-activated NtrC receiver domain." Biochemistry 42.30 (2003): 9081-9090.
PMID
12885241
Source
scival
Published In
Biochemistry
Volume
42
Issue
30
Publish Date
2003
Start Page
9081
End Page
9090
DOI
10.1021/bi0273866

Regulation of the transcriptional activator NtrC1: Structural studies of the regulatory and AAA+ ATPase domains

Transcription by σ54 RNA polymerase depends on activators that contain ATPase domains of the AAA+ class. These activators, which are often response regulators of two-component signal transduction systems, remodel the polymerase so that it can form open complexes at promoters. Here, we report the first crystal structures of the ATPase domain of an activator, the NtrC1 protein from the extreme thermophile Aquifex aeolicus. This domain alone, which is active, crystallized as a ring-shaped heptamer. The protein carrying both the ATPase and adjacent receiver domains, which is inactive, crystallized as a dimer. In the inactive dimer, one residue needed for catalysis is far from the active site, and extensive contacts among the domains prevent oligomerization of the ATPase domain. Oligomerization, which completes the active site, depends on surfaces that are buried in the dimer, and hence, on a rearrangement of the receiver domains upon phosphorylation. A motif in the ATPase domain known to be critical for coupling energy to remodeling of polymerase forms a novel loop that projects from the middle of an α helix. The extended, structured loops from the subunits of the heptamer localize to a pore in the center of the ring and form a surface that could contact σ54.

Authors
Lee, S-Y; Torre, ADL; Yan, D; Kustu, S; Nixon, BT; Wemmer, DE
MLA Citation
Lee, S-Y, Torre, ADL, Yan, D, Kustu, S, Nixon, BT, and Wemmer, DE. "Regulation of the transcriptional activator NtrC1: Structural studies of the regulatory and AAA+ ATPase domains." Genes and Development 17.20 (2003): 2552-2563.
PMID
14561776
Source
scival
Published In
Genes and Development
Volume
17
Issue
20
Publish Date
2003
Start Page
2552
End Page
2563
DOI
10.1101/gad.1125603

The NMR solution structure of BeF3--activated Spo0F reveals the conformational switch in a phosphorelay system

Two-component systems, which are comprised of a single histidine-aspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidine-aspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF3--activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF3- activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators. © 2003 Elsevier Ltd. All rights reserved.

Authors
Gardino, AK; Volkman, BF; Cho, HS; Lee, S-Y; Wemmer, DE; Kern, D
MLA Citation
Gardino, AK, Volkman, BF, Cho, HS, Lee, S-Y, Wemmer, DE, and Kern, D. "The NMR solution structure of BeF3--activated Spo0F reveals the conformational switch in a phosphorelay system." Journal of Molecular Biology 331.1 (2003): 245-254.
PMID
12875849
Source
scival
Published In
Journal of Molecular Biology
Volume
331
Issue
1
Publish Date
2003
Start Page
245
End Page
254
DOI
10.1016/S0022-2836(03)00733-2

Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy

Xenon-binding sites in proteins have led to a number of applications of xenon in biochemical and structural studies. Here we further develop the utility of 129Xe NMR in characterizing specific xenon-protein interactions. The sensitivity of the 129Xe chemical shift to its local environment and the intense signals attainable by optical pumping make xenon a useful NMR reporter of its own interactions with proteins. A method for detecting specific xenon-binding interactions by analysis of 129Xe chemical shift data is illustrated using the maltose binding protein (MBP) from Escherichia coli as an example. The crystal structure of MBP in the presence of 8 atm of xenon confirms the binding site determined from NMR data. Changes in the structure of the xenon-binding cavity upon the binding of maltose by the protein can account for the sensitivity of the 129Xe chemical shift to MBP conformation. 129Xe NMR data for xenon in solution with a number of cavity containing phage T4 lysozyme mutants show that xenon can report on cavity structure. In particular, a correlation exists between cavity size and the binding-induced 129Xe chemical shift. Further applications of 129Xe NMR to biochemical assays, including the screening of proteins for xenon binding for crystallography are considered. © 2002 Elsevier Science Ltd. All rights reserved.

Authors
Rubin, SM; Lee, S-Y; Ruiz, EJ; Pines, A; Wemmer, DE
MLA Citation
Rubin, SM, Lee, S-Y, Ruiz, EJ, Pines, A, and Wemmer, DE. "Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy." Journal of Molecular Biology 322.2 (2002): 425-440.
PMID
12217701
Source
scival
Published In
Journal of Molecular Biology
Volume
322
Issue
2
Publish Date
2002
Start Page
425
End Page
440
DOI
10.1016/S0022-2836(02)00739-8

Crystal structure of activated CheY: Comparison with other activated receiver domains

The crystal structure of BeF3--activated CheY, with manganese in the magnesium binding site, was determined at 2.4-Å resolution. BeF3- bonds to Asp57, the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr 87 and Lys109, respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF3-, the side chain oxygens of Asp13 and Asp57, the carbonyl oxygen of Asn59, and two water molecules. All of the active site interactions seen for BeF3--CheY are also observed in P-Spo0Ar. Thus, BeF3- activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr 106 is found buried within a hydrophobic pocket formed by β-strand β4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu89. This hydrogen bond appears to stabilize the active conformation of the β4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the β4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJr. A common feature in the active sites of BeF3--CheY, P-Spo0Ar, P-FixJr, and phosphono-CheY is a salt bridge between Lys109 Nζ and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr87 and Tyr106 (Thr-Tyr coupling), formation of the Lys109-PO3- salt bridge is directly involved in the activation of receiver domains generally.

Authors
Lee, S-Y; Cho, HS; Pelton, JG; Yan, D; Berry, EA; Wemmer, DE
MLA Citation
Lee, S-Y, Cho, HS, Pelton, JG, Yan, D, Berry, EA, and Wemmer, DE. "Crystal structure of activated CheY: Comparison with other activated receiver domains." Journal of Biological Chemistry 276.19 (2001): 16425-16431.
PMID
11279165
Source
scival
Published In
Journal of Biological Chemistry
Volume
276
Issue
19
Publish Date
2001
Start Page
16425
End Page
16431
DOI
10.1074/jbc.M101002200

Crystal structure of an activated response regulator bound to its target

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the β4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-β5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

Authors
Lee, S-Y; Cho, HS; Pelton, JG; Yan, D; Henderson, RK; King, DS; Huang, L-S; Kustu, S; Berry, EA; Wemmer, DE
MLA Citation
Lee, S-Y, Cho, HS, Pelton, JG, Yan, D, Henderson, RK, King, DS, Huang, L-S, Kustu, S, Berry, EA, and Wemmer, DE. "Crystal structure of an activated response regulator bound to its target." Nature Structural Biology 8.1 (2001): 52-56.
PMID
11135671
Source
scival
Published In
Nature Structural Biology
Volume
8
Issue
1
Publish Date
2001
Start Page
52
End Page
56
DOI
10.1038/83053

NMR structure of activated CheY

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF3/-) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(±0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF3/-, stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of β4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains. (C) 2000 Academic Press.

Authors
Cho, HS; Lee, S-Y; Yan, D; Pan, X; Parkinson, JS; Kustu, S; Wemmer, DE; Pelton, JG
MLA Citation
Cho, HS, Lee, S-Y, Yan, D, Pan, X, Parkinson, JS, Kustu, S, Wemmer, DE, and Pelton, JG. "NMR structure of activated CheY." Journal of Molecular Biology 297.3 (2000): 543-551.
PMID
10731410
Source
scival
Published In
Journal of Molecular Biology
Volume
297
Issue
3
Publish Date
2000
Start Page
543
End Page
551
DOI
10.1006/jmbi.2000.3595

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

Authors
Yan, D; Cho, HS; Hastings, CA; Igo, MM; Lee, S-Y; Pelton, JG; Stewart, V; Wemmer, DE; Kustu, S
MLA Citation
Yan, D, Cho, HS, Hastings, CA, Igo, MM, Lee, S-Y, Pelton, JG, Stewart, V, Wemmer, DE, and Kustu, S. "Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators." Proceedings of the National Academy of Sciences of the United States of America 96.26 (1999): 14789-14794.
PMID
10611291
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
96
Issue
26
Publish Date
1999
Start Page
14789
End Page
14794
DOI
10.1073/pnas.96.26.14789

Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable α-helix spanning residues Phe11- Ile26 and an antiparallel β-sheet formed by residues 2-5, 36-38, 41-47, 54- 64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding β-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands β2 and β3 are connected by a small bulge comprising residues Ile38- Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures (<SA>(k)) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 Å for secondary structural regions and 0.70 Å for backbone atoms excluding two loop regions. The average restraint energy- minimized (REM) structure exhibited root-mean-square deviations of 1.19 Å for backbone atoms and 0.85 Å for backbone atoms excluding two loop regions with respect to 20 <SA>(k) structures. The solution structure of SCM revealed that the long α-helix was folded into the concave side of a six-stranded antiparallel β-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.

Authors
Lee, S-Y; Lee, J-H; Chang, H-J; Cho, JM; Jung, J-W; Lee, W
MLA Citation
Lee, S-Y, Lee, J-H, Chang, H-J, Cho, JM, Jung, J-W, and Lee, W. "Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations." Biochemistry 38.8 (1999): 2340-2346.
PMID
10029527
Source
scival
Published In
Biochemistry
Volume
38
Issue
8
Publish Date
1999
Start Page
2340
End Page
2346
DOI
10.1021/bi9822731
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