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In the Peptide-Loading Complex Mechanistic Basis for Epitope Mechanistic Basis for Epitope Proofreading in the Peptide-Loading Complex Gerda Fleischmann, Olivier Fisette, Christoph Thomas, Ralph Wieneke, Franz Tumulka, Clemens Schneeweiss, This information is current as Sebastian Springer, Lars V. Schäfer and Robert Tampé of September 24, 2021. J Immunol 2015; 195:4503-4513; Prepublished online 28 September 2015; doi: 10.4049/jimmunol.1501515 http://www.jimmunol.org/content/195/9/4503 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/09/28/jimmunol.150151 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 59 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/195/9/4503.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average 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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Mechanistic Basis for Epitope Proofreading in the Peptide-Loading Complex Gerda Fleischmann,* Olivier Fisette,† Christoph Thomas,* Ralph Wieneke,* Franz Tumulka,* Clemens Schneeweiss,‡ Sebastian Springer,‡ Lars V. Scha¨fer,† and Robert Tampe´* The peptide-loading complex plays a pivotal role in Ag processing and is thus central to the efficient immune recognition of virally and malignantly transformed cells. The underlying mechanism by which MHC class I (MHC I) molecules sample immunodominant peptide epitopes, however, remains poorly understood. In this article, we delineate the interaction between tapasin (Tsn) and MHC I molecules. We followed the process of peptide editing in real time after ultra-fast photoconversion to pseudoempty MHC I mol- ecules. Tsn discriminates between MHC I loaded with optimal and MHC I bound to suboptimal cargo. This differential interaction is key to understanding the kinetics of epitope proofreading. To elucidate the underlying mechanism at the atomic level, we modeled Downloaded from the Tsn/MHC I complex using all-atom molecular dynamics simulations. We present a catalytic working cycle, in which Tsn binds to MHC I with suboptimal cargo and thereby adjusts the energy landscape in favor of MHC I complexes with immunodominant epitopes. The Journal of Immunology, 2015, 195: 4503–4513. ytotoxic T lymphocytes recognize virally or malignantly critical role in Ag processing was confirmed in Tsn2/2 mice, transformed cells via antigenic peptide epitopes presented where a drastic reduction in MHC I cell-surface expression was http://www.jimmunol.org/ C onMHCclassI(MHCI)molecules.SelectionofMHCI observed when compared with Tsn-proficient cells (8, 9). Notably, loaded with immunodominant epitopes requires a sophisticated soluble Tsn, which lacks the transmembrane domain and cytosolic interplay of various factors including the transporter associated tail, can partly complement MHC I loading and cell-surface ex- with Ag processing (TAP) and tapasin (Tsn) as key players, as pression (10). Tsn has been shown to play a key role in catalyzing well as auxiliary chaperones such as calreticulin and the thiol- peptide loading of MHC I (11) in a process called peptide opti- dependent oxidoreductase ERp57 that is disulfide-linked to Tsn. mization (12). The Tsn-ERp57 conjugate acts as a scaffold for Together, they comprise the endoplasmic reticulum (ER)-resident other PLC components, especially in recruiting and stabilizing peptide-loading complex (PLC) centered on the TAP complex (1, peptide-receptive MHC I molecules (13). In addition, Tsn binds to by guest on September 24, 2021 2). The molecular events during MHC I peptide loading are still TAP, thus bridging peptide donor and acceptor (14, 15). The not well defined. In particular, our understanding of MHC I function of ERp57 is mainly a structural one, promoting the Tsn– proofreading through direct contact between Tsn and MHC I still MHC I interaction (3, 13). The Tsn–MHC interaction is thought needs further development. X-ray structures of a range of MHC I to be mediated by two conserved ER-lumenal interfaces (3, 16–19). molecules and the Tsn-ERp57 conjugate have been described MHC I alleles differ in their dependence on Tsn with respect previously (3, 4); the architecture of the Tsn/MHC I complex to the acquisition of peptides (20–22). Notably, the HLA alleles within the PLC, however, remains to be determined. B*44:02 and B*44:05 differ only by a single residue at position The crucial function of Tsn in MHC I loading was first described 116, yet they diverge markedly in their dependence on Tsn: in studies using the Tsn-deficient human cell line 721.220 (5–7). Its B*44:02 carries an aspartate and is Tsn dependent, whereas B*44:05 contains a tyrosine and is Tsn independent. It has been *Institute of Biochemistry, Biocenter, Goethe University Frankfurt, 60438 Frankfurt, suggested that Tsn widens the peptide-binding pocket and gen- † Germany; Lehrstuhl fur€ Theoretische Chemie, Ruhr-University Bochum, 44780 erates an energy barrier, which allows only the binding of high- Bochum, Germany; and ‡Molecular Life Science, Jacobs University Bremen, 28759 Bremen, Germany affinity peptides, thereby disengaging Tsn (23). By analyzing b Received for publication July 29, 2015. Accepted for publication August 31, 2015. peptide loading onto the MHC I allele H-2K using isolated This work was supported by German Research Foundation Project SFB 807 “Mem- microsomes, Tsn was shown to increase dissociation rates of brane Transport and Communication” (to R.T. and L.V.S.), Cluster of Excellence peptides and thus to reduce the concentration of unstable MHC I Ruhr Explores Solvation EXC 1069 (to L.V.S.), Graduate School Complex Scenarios complexes with low-affinity peptides (24). of Light-Control (to R.T.), Emmy Noether Grant SCHA1574/3-1 (to L.V.S.), and a European Molecular Biology Organization short-term fellowship (to C.S.). Because an atomic-level picture of the structure and dynamics Address correspondence and reprint requests to Prof. Robert Tampe´, Institute of of the complex between MHC I and Tsn was lacking so far, Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue-Strasse 9, the mechanistic principle of peptide optimization has remained 60438 Frankfurt am Main, Germany. E-mail address: [email protected] enigmatic (25). We followed peptide editing in real time in the The online version of this article contains supplemental material. presence and absence of Tsn and determined the kinetics as well Abbreviations used in this article: AF633, Alexa Fluor 633; BirA, biotin ligase A; as thermodynamics of the interaction between Tsn and MHC I. dNA, dimeric NeutrAvidin; ER, endoplasmic reticulum; b2-m, b2-microglobulin; MD, molecular dynamics; MHC I, MHC class I; PLC, peptide-loading complex; The process of peptide optimization was synchronized by a pho- SEC-MALLS, size-exclusion chromatography multiangle laser light scattering; toreaction, converting a high-affinity epitope into a low-affinity SPR, surface plasmon resonance; TAP, transporter associated with Ag processing; cargo. We show that Tsn accelerates the dissociation rate of low- Tn6, Tsn variant 6; Tsn, tapasin. and medium-affinity (suboptimal) peptide epitopes. Moreover, the Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 differential binding of Tsn to peptide-loaded and peptide-deficient www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501515 4504 MHC I PROOFREADING MHC I was observed, which is crucial for peptide editing and se- gel filtration using a Superdex 200 (GE Healthcare) in HEPES-E buffer. lection of immunodominant epitopes. To unravel the underlying The Tsn-ERp57 conjugate was concentrated using Amicon Ultra-15 mechanism at the atomic level, we obtained the structure of the Tsn/ devices (Millipore). MHC I complex from multimicrosecond all-atom molecular dy- Assembly of MHC I/Tsn-ERp57/dimeric NeutrAvidin namics (MD) simulations. Tsn and the Ag peptide compete for complexes opening/closing the MHC I binding groove, thereby modulating the Biotin ligase A (BirA) (pET21-BirA; Addgene) was expressed in E. coli affinity of the Tsn/MHC I complex. and purified as described previously (28). B*44:02 (30 mM) and Tsn- ERp57 (30 mM) were incubated with BirA (1 mM) in BirA reaction Materials and Methods buffer (50 mM bicine, pH 8.3, 10 mM biotin, 1 mM MgATP). After 2 h at Retrovirus and stable cell line expressing single-chain HLA- 30˚C, free biotin was removed by rapid gel filtration (MicroSpin G25; Bio- B*44:02 Rad). The biotinylation of the two components was compared by immu- noblotting using streptactin-conjugated HRP. Biotinylated B*44:02 (10 The DNA sequence coding for HLA-B*44:02 (aa 25-298) was PCR- mM) was first titrated to equimolar amounts of dimeric NeutrAvidin (dNA, amplified from pCSB53 using the forward primer (59-CCTTAAT- Thermo Scientific). Subsequently, twice the amount of Tsn-ERp57 (20 TAACGGCTCCCACTCCATGAGGTATTT-39) and the reverse primer (59- mM, biotinylation efficiency was lower in comparison with B*44:02) was CTGCACCGGTCCATCTCAGGGTGAGGGGCTTC-39). The resulting added and incubated at 4˚C for 30 min in HEPES-E buffer to yield construct was cloned into a plasmid containing a GFP gene after an in- a stoichiometric B*44:02/Tsn-ERp57/dNA complex. The monodispersity ternal ribosomal entry site for selection, kindly provided by A. Townsend of the complexes was analyzed in HEPES-E buffer pH 7.0 using size- (Oxford University).
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