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Previously Hidden Dynamics at the TCR–Peptide–MHC Interface Revealed Previously Hidden Dynamics at the TCR− Peptide−MHC Interface Revealed James Fodor, Blake T. Riley, Natalie A. Borg and Ashley M. Buckle This information is current as of September 25, 2021. J Immunol published online 4 May 2018 http://www.jimmunol.org/content/early/2018/05/04/jimmun ol.1800315 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/05/04/jimmunol.180031 Material 5.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 4, 2018, doi:10.4049/jimmunol.1800315 The Journal of Immunology Previously Hidden Dynamics at the TCR–Peptide–MHC Interface Revealed James Fodor, Blake T. Riley, Natalie A. Borg,1 and Ashley M. Buckle1 A structural characterization of the interaction between ab TCRs and cognate peptide–MHC (pMHC) is central to understanding adaptive T cell–mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby reveal- ing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship Downloaded from between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR–pMHC interactions. The http://www.jimmunol.org/ Journal of Immunology, 2018, 200: 000–000. ytotoxic T cells express on their cell surface ab TCRs, dynamics and degree of flexibility of the system in solution (1–3). which engage endogenous peptide Ags from infected or Given that protein flexibility is inextricably linked to protein func- C malignant cells that are displayed by MHC proteins. tion, consideration of protein dynamics is essential to achieve a Productive engagement of the TCR with the peptide–MHC full understanding of pMHC and TCR–pMHC binding. Recent (pMHC) leads to T cell activation and intracellular signal trans- years have seen an increasing use of atomistic molecular dy- duction via the CD3 signaling subunits. This TCR–pMHC molecular namics (MD) simulations, which can probe the conformational recognition event is key to the cell-mediated adaptive immune re- flexibility of pMHC systems by computing iterative numerical by guest on September 25, 2021 sponse and has therefore been widely studied using x-ray crystal- solutions of Newton’s equations of motion as the system evolves lography. These studies have focused on the conformation of the over time (4). Unfortunately, the heavy computational demands peptide when bound to the MHC and the specific interactions be- of MD limit the technique to examination of relatively short time tween peptide and MHC side chains, with or without bound TCR. spans (usually less than a microsecond) and thereby exclude many Although providing insights into the key features of TCR–pMHC larger motions that occur over longer, more biologically relevant recognition, the detailed examination of a single static structure timescales. Results of MD are also somewhat difficult to validate by x-ray crystallography reveals little about the conformational given the known imperfections of existing force fields (5–7). Ensemble x-ray structure refinement is a refinement technique that can be employed to extend the interpretation of crystallographic Infection and Immunity Program, Biomedicine Discovery Institute, Monash Univer- data and obtain information about protein dynamics without the sity, Clayton, Victoria 3800, Australia; and Department of Biochemistry and Molec- necessity of running expensive MD simulations (8). Instead of the ular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia usual practice of refining against a set of crystallographic reflections 1 to produce a single protein structure, ensemble refinement combines N.A.B. and A.M.B. are joint senior authors. the experimentally derived reflection data with the results of a short, ORCIDs: 0000-0003-2176-0503 (B.T.R.); 0000-0002-8677-9056 (N.A.B.); 0000- 0003-2943-9044 (A.M.B.). steered MD simulation to produce an ensemble of similar but dis- Received for publication March 6, 2018. Accepted for publication April 10, 2018. tinct conformations of the protein. The structural diversity observed within the resulting ensemble thus provides information about the This work was supported by grants from the National Health and Medical Research Council and the Australian Research Council (ARC). N.A.B. is funded by an ARC conformational flexibility of the protein and, in many cases, im- Future Fellowship (110100223). This work was supported by the Multi-Modal Aus- proves the agreement between the structure and data as measured by tralian Sciences Imaging and Visualisation Environment. the crystallographic free R-factor (Rfree). Notable recent applica- Address correspondence and reprint requests to Prof. Ashley M. Buckle and tions of this method include an investigation of the conformational Dr. Natalie A. Borg, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia. landscape of an insect carboxylesterase protein over the course of its E-mail addresses: [email protected] (A.M.B.) and natalie.borg@monash. catalytic cycle (9) and the discovery of a self-inhibition mechanism edu (N.A.B.) mediating the transition between active and inactive protein of The online version of this article contains supplemental material. human factor D (10). Ensemble refinement is therefore a technique Abbreviations used in this article: MD, molecular dynamics; NP, nucleoprotein; PDB, for extracting dynamical information already present in x-ray re- Protein Data Bank; pMHC, peptide–MHC; R , free R-factor. free flection data but overlooked in the process of producing a single Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 static structural model. As such, we hypothesized that when applied www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800315 2 TCR–pMHC DYNAMICS to pMHC and TCR–pMHC systems, ensemble refinement would square fluctuation ranking as being the most relevant for testing uncover evidence of molecular motion that has hitherto been hid- our hypothesis. For each of these systems, we compared our re- den. We further hypothesized that the dynamic information revealed sults in detail to those reported in each original publication. would in many cases significantly enhance existing single-structure In comparing our ensemble results with the analyses reported in analyses of these systems. In this study, we test these hypotheses by the original papers, we found it necessary to run an additional 25 applying ensemble refinement to investigate protein dynamics at the ensembles to properly interpret our ensemble results, thus raising the TCR–pMHC interface. total number of systems for which ensembles were calculated to 309. Given space limitations, from these 30 systems we selected 11 il- Materials and Methods lustrative cases in which our ensemble results provided the greatest Computational resources functional insights pertinent to the claims made in the original lit- erature. Because in some cases the original study compared multiple Calculations, modeling, and simulations were performed on a range of pMHC systems, these 11 examples included a total of 20 distinct computing resources: ORCHARD 800 core x86 cluster (Monash Univer- sity; x-ray ensemble refinement) and Multi-Modal Australian Sciences PDB structures, all of which are summarized in Table I. In our Imaging and Visualisation Environment
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