Browsing by Subject "Crystallography, X-Ray"

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Open Access
A kinesin motor in a force-producing conformation.
(BMC Struct Biol, 2010-07-05) Heuston, Elisabeth; Bronner, C Eric; Kull, F Jon; Endow, Sharyn A
BACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
Unknown
Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.
(Nature, 2013-04-25) Liao, Hua-Xin; Lynch, Rebecca; Zhou, Tongqing; Gao, Feng; Alam, S Munir; Boyd, Scott D; Fire, Andrew Z; Roskin, Krishna M; Schramm, Chaim A; Zhang, Zhenhai; Zhu, Jiang; Shapiro, Lawrence; NISC Comparative Sequencing Program; Mullikin, James C; Gnanakaran, S; Hraber, Peter; Wiehe, Kevin; Kelsoe, Garnett; Yang, Guang; Xia, Shi-Mao; Montefiori, David C; Parks, Robert; Lloyd, Krissey E; Scearce, Richard M; Soderberg, Kelly A; Cohen, Myron; Kamanga, Gift; Louder, Mark K; Tran, Lillian M; Chen, Yue; Cai, Fangping; Chen, Sheri; Moquin, Stephanie; Du, Xiulian; Joyce, M Gordon; Srivatsan, Sanjay; Zhang, Baoshan; Zheng, Anqi; Shaw, George M; Hahn, Beatrice H; Kepler, Thomas B; Korber, Bette TM; Kwong, Peter D; Mascola, John R; Haynes, Barton F
Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.
Open Access
Competition between monomeric and dimeric crystals in schematic models for globular proteins.
(J Phys Chem B, 2014-07-17) Fusco, Diana; Charbonneau, Patrick
Advances in experimental techniques and in theoretical models have improved our understanding of protein crystallization. However, they have also left open questions regarding the protein phase behavior and self-assembly kinetics, such as why (nearly) identical crystallization conditions can sometimes result in the formation of different crystal forms. Here, we develop a patchy particle model with competing sets of patches that provides a microscopic explanation of this phenomenon. We identify different regimes in which one or two crystal forms can coexist with a low-density fluid. Using analytical approximations, we extend our findings to different crystal phases, providing a general framework for treating protein crystallization when multiple crystal forms compete. Our results also suggest different experimental routes for targeting a specific crystal form, and for reducing the dynamical competition between the two forms, thus facilitating protein crystal assembly.
Open Access
Computational Methods for RNA Structure Validation and Improvement.
(Methods Enzymol, 2015) Jain, Swati; Richardson, David C; Richardson, Jane S
With increasing recognition of the roles RNA molecules and RNA/protein complexes play in an unexpected variety of biological processes, understanding of RNA structure-function relationships is of high current importance. To make clean biological interpretations from three-dimensional structures, it is imperative to have high-quality, accurate RNA crystal structures available, and the community has thoroughly embraced that goal. However, due to the many degrees of freedom inherent in RNA structure (especially for the backbone), it is a significant challenge to succeed in building accurate experimental models for RNA structures. This chapter describes the tools and techniques our research group and our collaborators have developed over the years to help RNA structural biologists both evaluate and achieve better accuracy. Expert analysis of large, high-resolution, quality-conscious RNA datasets provides the fundamental information that enables automated methods for robust and efficient error diagnosis in validating RNA structures at all resolutions. The even more crucial goal of correcting the diagnosed outliers has steadily developed toward highly effective, computationally based techniques. Automation enables solving complex issues in large RNA structures, but cannot circumvent the need for thoughtful examination of local details, and so we also provide some guidance for interpreting and acting on the results of current structure validation for RNA.
Open Access
DNA mismatches reveal conformational penalties in protein-DNA recognition.
(Nature, 2020-11) Afek, Ariel; Shi, Honglue; Rangadurai, Atul; Sahay, Harshit; Senitzki, Alon; Xhani, Suela; Fang, Mimi; Salinas, Raul; Mielko, Zachery; Pufall, Miles A; Poon, Gregory MK; Haran, Tali E; Schumacher, Maria A; Al-Hashimi, Hashim M; Gordân, Raluca
Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood^1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble^3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell^10,11.
Open Access
Drug design from the cryptic inhibitor envelope.
(Nat Commun, 2016-02-25) Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J; Zhou, Pei
Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.
Open Access
Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.
(PLoS One, 2010-09-09) Wu, Shenping; Liu, Jun; Reedy, Mary C; Tregear, Richard T; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E; Reedy, Michael K; Taylor, Kenneth A
BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.
Open Access
FtsZ filament capping by MciZ, a developmental regulator of bacterial division.
(Proceedings of the National Academy of Sciences of the United States of America, 2015-04-06) Bisson-Filho, Alexandre W; Discola, Karen F; Castellen, Patrícia; Blasios, Valdir; Martins, Alexandre; Sforça, Maurício L; Garcia, Wanius; Zeri, Ana Carolina M; Erickson, Harold P; Dessen, Andréa; Gueiros-Filho, Frederico J
Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.
Open Access
Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.
(Nature communications, 2019-09) Juvvadi, Praveen R; Fox, David; Bobay, Benjamin G; Hoy, Michael J; Gobeil, Sophie MC; Venters, Ronald A; Chang, Zanetta; Lin, Jackie J; Averette, Anna Floyd; Cole, D Christopher; Barrington, Blake C; Wheaton, Joshua D; Ciofani, Maria; Trzoss, Michael; Li, Xiaoming; Lee, Soo Chan; Chen, Ying-Lien; Mutz, Mitchell; Spicer, Leonard D; Schumacher, Maria A; Heitman, Joseph; Steinbach, William J
Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.
Open Access
High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP.
(Acta crystallographica. Section F, Structural biology communications, 2020-02-05) Schumacher, Maria A; Ohashi, Tomoo; Corbin, Lauren; Erickson, Harold P
Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.
Open Access
Infrared Spectroscopic Observation of a G-C+ Hoogsteen Base Pair in the DNA:TATA-Box Binding Protein Complex Under Solution Conditions.
(Angewandte Chemie (International ed. in English), 2019-08) Stelling, Allison L; Liu, Amy Y; Zeng, Wenjie; Salinas, Raul; Schumacher, Maria A; Al-Hashimi, Hashim M
Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson-Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X-ray structures of protein-DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson-Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X-ray crystallography due to ambiguous electron density and by solution-state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution-state observation of a Hoogsteen (G-C+ ) bp in a DNA:protein complex under solution conditions with specific application to DNA-bound TATA-box binding protein. These results support a previous assignment of a G-C+ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.
Open Access
Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor.
(Nature, 2010-11) Sheard, Laura B; Tan, Xu; Mao, Haibin; Withers, John; Ben-Nissan, Gili; Hinds, Thomas R; Kobayashi, Yuichi; Hsu, Fong-Fu; Sharon, Michal; Browse, John; He, Sheng Yang; Rizo, Josep; Howe, Gregg A; Zheng, Ning
Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.
Restricted
Multiscale conformational heterogeneity in staphylococcal protein a: possible determinant of functional plasticity.
(Structure, 2014-10-07) Deis, Lindsay N; Pemble, Charles W; Qi, Yang; Hagarman, Andrew; Richardson, David C; Richardson, Jane S; Oas, Terrence G
The Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies. SpA includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner have apparently been unsuccessful. There have also been no previous structures of tandem domains. Here we report the high-resolution crystal structures of a single C domain, and of two B domains connected by the conserved linker. Both structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in side chain conformational networks and contacting protein interfaces. This represents the kind of structural plasticity that could enable SpA to bind multiple partners.
Open Access
New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey.
(Nucleic Acids Res, 2015-04-20) Zhou, H; Hintze, BJ; Kimsey, IJ; Sathyamoorthy, B; Yang, S; Richardson, JS; Al-Hashimi, HM
Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson-Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1'-C1' distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1'-C1' distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5'-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA.
Open Access
Statistical analysis of crystallization database links protein physico-chemical features with crystallization mechanisms.
(PLoS One, 2014) Fusco, Diana; Barnum, Timothy J; Bruno, Andrew E; Luft, Joseph R; Snell, Edward H; Mukherjee, Sayan; Charbonneau, Patrick
X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.
Open Access
Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase.
(Cell reports, 2015-06-18) Choy, Meng S; Yusoff, Permeen; Lee, Irene C; Newton, Jocelyn C; Goh, Catherine W; Page, Rebecca; Shenolikar, Shirish; Peti, Wolfgang
The attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.
Open Access
Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling.
(Nature, 2015-09) Zhang, Feng; Yao, Jian; Ke, Jiyuan; Zhang, Li; Lam, Vinh Q; Xin, Xiu-Fang; Zhou, X Edward; Chen, Jian; Brunzelle, Joseph; Griffin, Patrick R; Zhou, Mingguo; Xu, H Eric; Melcher, Karsten; He, Sheng Yang
The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1-JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.
Open Access
Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9.
(Nature, 2011-11-23) McLellan, JS; Pancera, M; Carrico, C; Gorman, J; Julien, JP; Khayat, R; Louder, R; Pejchal, R; Sastry, M; Dai, K; O'Dell, S; Patel, N; Shahzad ul Hussan, S; Yang, Y; Zhang, B; Zhou, T; Zhu, J; Boyington, JC; Chuang, GY; Diwanji, D; Georgiev, I; Kwon, YD; Lee, D; Louder, MK; Moquin, S; Schmidt, SD; Yang, ZY; Bonsignori, M; Crump, JA; Kapiga, SH; Sam, NE; Haynes, BF; Burton, DR; Koff, WC; Walker, LM; Phogat, S; Wyatt, R; Orwenyo, J; Wang, LX; Arthos, J; Bewley, CA; Mascola, JR; Nabel, GJ; Schief, WR; Ward, AB; Wilson, IA; Kwong, PD
Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.
Open Access
Suppression of conformational heterogeneity at a protein-protein interface.
(Proc Natl Acad Sci U S A, 2015-07-21) Deis, Lindsay N; Wu, Qinglin; Wang, You; Qi, Yang; Daniels, Kyle G; Zhou, Pei; Oas, Terrence G
Staphylococcal protein A (SpA) is an important virulence factor from Staphylococcus aureus responsible for the bacterium's evasion of the host immune system. SpA includes five small three-helix-bundle domains that can each bind with high affinity to many host proteins such as antibodies. The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure was not properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. Here we report the 2.3-Å structure of a fully folded SpA domain in complex with Fc. Our structure indicates that there are extensive structural rearrangements necessary for binding Fc, including a general reduction in SpA conformational heterogeneity, freezing out of polyrotameric interfacial residues, and displacement of a SpA side chain by an Fc side chain in a molecular-recognition pocket. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur when SpA binds its multiple binding partners. Suppression of conformational heterogeneity may be an important structural paradigm in functionally plastic proteins.
Open Access
The importance of residue-level filtering and the Top2018 best-parts dataset of high-quality protein residues.
(Protein science : a publication of the Protein Society, 2022-01) Williams, Christopher J; Richardson, David C; Richardson, Jane S
We have curated a high-quality, "best-parts" reference dataset of about 3 million protein residues in about 15,000 PDB-format coordinate files, each containing only residues with good electron density support for a physically acceptable model conformation. The resulting prefiltered data typically contain the entire core of each chain, in quite long continuous fragments. Each reference file is a single protein chain, and the total set of files were selected for low redundancy, high resolution, good MolProbity score, and other chain-level criteria. Then each residue was critically tested for adequate local map quality to firmly support its conformation, which must also be free of serious clashes or covalent-geometry outliers. The resulting Top2018 prefiltered datasets have been released on the Zenodo online web service and are freely available for all uses under a Creative Commons license. Currently, one dataset is residue filtered on main chain plus Cβ atoms, and a second dataset is full-residue filtered; each is available at four different sequence-identity levels. Here, we illustrate both statistics and examples that show the beneficial consequences of residue-level filtering. That process is necessary because even the best of structures contain a few highly disordered local regions with poor density and low-confidence conformations that should not be included in reference data. Therefore, the open distribution of these very large, prefiltered reference datasets constitutes a notable advance for structural bioinformatics and the fields that depend upon it.