Bidirectional binding of invariant chain peptides to an MHC class II molecule Sebastian Günthera,b,1, Andreas Schlundta,1, Jana Stichta, Yvette Roskeb, Udo Heinemannb, Karl-Heinz Wiesmüllerc, Günther Jungc, Kirsten Falkb, Olaf Rötzschkeb,d, and Christian Freunda,2 aLeibniz-Institute for Molecular Pharmacology and Freie Universität Berlin, 13125 Berlin, Germany; bMax-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany; cEMC Microcollections GmbH, D-72070 Tübingen, Germany; and dSingapore Immunology Network, Agency of Science, Technology and Research, Singapore 138648 Edited* by Emil R. Unanue, Washington University, St. Louis, MO, and approved November 2, 2010 (received for review September 30, 2010) T-cell recognition of peptides bound to MHC class II (MHCII) Here we determined the crystal structures of the human MHCII molecules is a central event in cell-mediated adaptive immunity. molecule HLA-DR1 in complex with two CLIP length variants The current paradigm holds that prebound class II-associated in- and observed a unique bidirectional binding mode. NMR analyses variant chain peptides (CLIP) and all subsequent antigens maintain by chemical shift mapping and spin label-induced relaxation en- a canonical orientation in the MHCII binding groove. Here we hancement of the uncatalyzed and HLA-DM edited reactions il- provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy lustrate the dynamic reorientation of the complex in solution. demonstrates that the interconversion from the canonical to the Surface charge distributions differ largely for the two MHC-CLIP inverse alignment is a dynamic process, and X-ray crystallography variants and result in T-cell epitopes with inverted polarity. shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst Results HLA-DM accelerates peptide reorientation and the exchange of Crystal Structures of HLA-DR1/CLIP Reveal Bidirectional Binding. We either canonically or inversely bound CLIP against antigenic peptide. determined the crystal structures of the human MHCII mole- Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally cule HLA-DR1 in complex with the two CLIP length variants and functionally intact antigen-presenting complex. CLIP106–120 and CLIP102–120 (see Fig. 1A for sequences; num- bering refers to the p35 form of invariant chain—see Methods IMMUNOLOGY bidirectional binding | antigen presentation | peptide loading for details). The latter exhibited a canonical peptide alignment showing all of the hallmarks of a conventional MHC–peptide mmune responses against extracellular pathogens rely on anti- complex [Figs. 1B (Right) and C, and 2A; Table S1]: the pocket P1 fi Igen presentation by MHC class II (MHCII) molecules (1). of the MHC molecule is occupied by the rst anchor residue near After endocytosis, exogenous proteins are proteolytically de- the N terminus of the peptide, and side chains 4, 6, and 9 protrude graded in endolysosomal vesicles that also harbor MHCII mole- into the corresponding surface depressions of the MHC binding cules, which at this point are bound to self-peptides originating groove. In contrast, the short CLIP variant was found to bind in an inverse orientation, with the C-terminal methionine accommo- from the invariant chain protein. HLA-DM catalyzed exchange of dated in P and the N-terminal methionine in P [Fig. 1B (Left) and class II-associated invariant chain peptides (CLIP) for pathogen- 1 9 C, and Table S1]. Whereas co-refolded HLA-DR1/CLIP – derived peptides of higher affinity then leads to the formation and 102 120 – crystallized at room temperature, crystals showing the inverted surface presentation of immunogenic MHCII peptide complexes orientation only grew at the same temperature when HLA-DR1 (2). However, evidence is mounting that CLIP function extends was refolded without any peptide and subsequently loaded with beyond its placeholder properties: stable MHCII–CLIP com- CLIP106–120. However, when changing the conditions to refolding plexes are up-regulated on dendritic cells upon maturation and HLA-DR1 in the presence of CLIP106–120 and crystallizing the modulate the activation of T cells specific for exogenous foreign complex at low temperatures (SI Materials and Methods)we antigen (3). The continued presentation of CLIP on the surface of could additionally determine the structure of canonically aligned antigen presenting cells requires the active maintenance of pe- CLIP106–120 bound to the MHC (Fig. S1). ripheral tolerance against self (4). MHC alleles susceptible to In-depth comparison of the three structures analyzed here autoimmune diseases, in contrast, are associated with low affini- reveals as a striking feature the preservation of a dense hydrogen ties for, and thus low surface levels of, CLIP (5). Taken together, bond network in both orientations (Fig. S2A). This particular the presence of the self-peptide CLIP on the cell surface seems to feature of peptide–MHC complexes is mediated by MHC side shape the T-cell repertoire in vivo, which, as observed for other chains that interact with the peptide backbone and, hence, is pep- peptides as well, has additionally been shown to be influenced by tide-sequence independent. Surprisingly, almost no rearrangement N-terminal length variations (6, 7). of MHC side chains is necessary to preserve the network in the The MHCII–peptide binding mode might account for these inverted orientation, and the pseudosymmetry of the polyproline observations. Peptides are assumed to bind in a conserved N- to type II helix ensures the availability of CO and NH peptide back- C-terminal orientation by two governing principles: (i) a set of hydrogen bonds between the peptide backbone and MHC side chains; and (ii) the geometrically and chemically favorable ac- Author contributions: S.G., A.S., J.S., O.R., and C.F. designed research; S.G., A.S., J.S., and fi fi Y.R. performed research; S.G., A.S., J.S., Y.R., U.H., K.-H.W., G.J., K.F., O.R., and C.F. commodation of four to ve peptide side chains by de ned MHC analyzed data; and S.G., A.S., J.S., and C.F. wrote the paper. – surface pockets (8). The observation that single peptide MHC The authors declare no conflict of interest. complexes can elicit functionally distinct immune responses has *This Direct Submission article had a prearranged editor. been attributed to distinct peptide binding registers (9, 10) or the Data deposition: The structures reported in this paper have been deposited in the Protein existence of different conformational isomers of the MHCII itself Data Bank database (accession nos. 3PDO, 3PGC, and 3PGD). (11, 12). However, because X-ray crystallography might not cap- 1S.G. and A.S. contributed equally to this work. ture the dynamic rearrangements that accompany alternative 2To whom correspondence should be addressed. E-mail: [email protected]. fi complex formation, the molecular basis for these ndings is still This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. poorly understood. 1073/pnas.1014708107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1014708107 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Crystal structures of HLA-DR1 bound to two CLIP length variants. (A) Amino acid sequences of CLIP variants used in this study. (B) Left: Crystal structure of HLA-DR1 bound to CLIP106–120 showing the peptide fragment in an atypical inverted orientation, as indicated by the arrow. Right: Analogous view onto the crystal structure of HLA-DR1 bound to CLIP102–120 in the canonical orientation. Both structures are shown as semitransparent surface (white) top view onto α1β1 domains, with the ribbon presentation embedded. The relative positions of the membrane distal domains are indicated. Peptides are displayed as sticks. (C) Upper: Lateral cut of the binding groove of HLA-DR1 (peptide removed), highlighting the antigen-binding pockets P1 and P9. Lower: 2Fo-Fc electron density maps (contoured at 1σ) of peptides derived from the crystal structure of HLA-DR1/CLIP106–120,flipped and HLA-DR1/CLIP102–120 are shown from the same per- spective inside the binding groove. Both peptides appear with two methionine side chains sequestered into the respective P1 and P9 pockets, as indicated. bone atoms as hydrogen bonding partners (Fig. S2 A and B and bilizing interface is missing. However, upon peptide inversion Table S2). In the reverse peptide orientation an additional H-bond these hydrogen bonds are reestablished (Fig. 2). Interestingly, the between the side chain carbonyl group of αN62 and the backbone only other available MHCII–CLIP structures are of longer CLIP NH group of P5 of the peptide can be formed (Fig. 2A and Fig. variants that are able to form these critical N-terminal hydro- S2A). The central threonine at P5 acts as point of symmetry, with gen bonds and display a canonical peptide orientation (13, 14). methionine occupying P1 and P9 and promiscuous binding of either alanine or proline at positions P4 and P6 (Fig. 1 A and C). Peptide Inversion Monitored by NMR Chemical Shift Analysis of HLA- A likely explanation for the observed peptide inversion is DR1/CLIP106–120. To ensure that bidirectional peptide alignment is revealed by a closer analysis of the hydrogen bond network not due to constraints imposed by the crystallization process, around P−2 of the MHC. At this position CLIP102–120 is engaged analysis of MHC-ligand complexes was carried out in solution. in three hydrogen bonds with the MHCII residues αF51 and The only spectroscopic method to yield such information at the αS53. In the canonical HLA-DR1/CLIP106–120 complex this sta- atomic level is NMR. Using an optimized refolding procedure 2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1014708107 Günther et al. Downloaded by guest on September 25, 2021 Fig. 3. Dynamic behavior of the HLA-DR1/CLIP106–120 complex. (A) Close-up view from crystal structures of HLA-DR1/CLIP106–120 with canonical (blue) and inverted (red) peptide orientation.