Bidirectional Binding of Invariant Chain Peptides to an MHC Class II Molecule

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; numbering 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. Residues Tyr78 and His81 located in the helix of β1 are in close contact to CLIP but experience different chemical environments in the two orientations. (B) Two regions of 1H-15N hetero- IMMUNOLOGY nuclear single quantum coherence spectral (HSQC) overlays of HLA-DR1(14Nα/ 15 Nβ)/CLIP106–120 reﬂect the peptide ﬂipping sensed by these two amino acids. The blue spectrum was measured directly after sample preparation by co- refolding, whereas the red one was recorded from the same sample after 120 h at 37 °C.

in isolation and loaded with CLIP106–120 a posteriori. In support, the NMR spectra of the latter assembly and the equilibrated co- refolded sample are fully superimposable (Fig. S4). Fig. 2. Lack of hydrogen bonds is the driving force in CLIP106–120 reorientation. (A–C) Magnifications of the region harboring the canonical pep- Spin-Labeled CLIP – Confirms the Inverted Orientation. The ’ 106 120 tide s N terminus of crystal structures of HLA-DR1 in complex with the chemical shift analysis is in full agreement with peptide inversion peptides CLIP – (A, blue), canonical CLIP – (B, violet) or CLIP – in 102 120 106 120 106 120 relative to the MHC binding groove, yet it only provides cir- the inverted orientation (C, red). For canonical CLIP106–120 three H-bonds from the HLA-DR1 α-chain residues Phe51 and Ser53 remain unsaturated, cumstantial evidence. To provide more direct confirmation of

whereas they are replaced in the inverted CLIP106–120 orientation (compare the noncanonical binding mode in solution, we performed NMR B and C). The peptide ligand is shown in yellow. experiments with CLIP106–120 carrying an N-terminally attached TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moiety (Fig. 4A). Atoms up to a distance of 15–20 Å of the unpaired electron (15), we obtained NMR spectra of high quality for several HLA- should experience enhanced relaxation leading to a clearly in- DR1/peptide complexes (Fig. S3 A and B) and assigned the 15 13 2 creased line-width of the corresponding NMR resonances. There- backbone chemical shifts for the N/ C/ H isotope-labeled fore, the TEMPO group should affect residues of the MHC β -chain of HLA-DR1/CLIP. molecule that accommodate the N-terminal methionine of the Interestingly, NMR spectra obtained from 15N-β-chain–labeled peptide. TEMPO-CLIP106–120 was loaded a posteriori to directly MHC molecules co-refolded with CLIP106–120 showed chemical obtain the thermodynamically preferred complex. We observed shift changes over time that exclusively map to the peptide- dramatic changes in NMR signal intensities for a single contigu- β binding 1 domain (Fig. S3 B and C). The initial spectrum ous region within the MHC molecule (Fig. 4B and C), comprising superimposed well with that obtained from the CLIP – com- 102 120 residues β54–68 that surround the P9 pocket of the antigen bind- plex, indicating that both assemblies indeed represent the same ing site (Fig. 4D). We conclude that the thermodynamically stable canonical binding mode observed in the crystal structures of MHCII–CLIP106–120 complex detected in solution contains a MHC refolded in the presence of peptide (Fig. S3A). Realignment peptide in the reverse orientation corresponding to the crystal of the peptide is then paralleled by time-dependent changes ob- structure shown in Fig. 1B (Left). served in the NMR spectra (Fig. S3B). Large NH backbone chemical shift differences (Fig. S3C) map to residues in the pep- HLA-DM–Catalyzed Peptide Inversion and Exchange Observed by tide binding groove, as for example βY78 and βH81, which ex- NMR. CLIP inversion from the kinetically trapped to the ther- perience a different chemical environment in the presence of modynamically more stable complex proceeds over a period of canonical or ﬂipped CLIP, as is seen in the corresponding crystal days at 37 °C, with a half-maximum inversion after ≈40 ± 6h.We structures (Fig. 3). Thus, this thermodynamically slightly more have to assume that CLIP cleaved from the invariant chain in the stable complex (Table S3) is suggested to be identical with the late endosome corresponds to the kinetically trapped variant crystallized inverted complex obtained from HLA-DR1 refolded found in our solution NMR studies (16). Peptide inversion would

Günther et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 then not occur in a physiologically relevant time frame. In vivo, however, peptide exchange is enhanced by the natural catalyst HLA-DM. It was therefore important to determine whether CLIP inversion could also be catalyzed. To further investigate this phenomenon, we capitalized on the finding that peptide reorientation was observed for the HLA-DR1/CLIP102–120 complex as well. This process was extremely slow and came to a halt at 50% inversion, indicating the same thermodynamic stability for both peptide alignments (Fig. S5 and Table S3). However, when adding equimolar amounts of purified HLA-DM to freshly refolded HLA-DR1 complexed with CLIP102–120, it catalyzed the 50% exchange within the experimental dead time of 30 min (Fig. 5A). We next asked whether both CLIP orientations would allow efficient exchange to a more tightly binding antigen. The influenza hem- agglutinin-derived peptide (HA306–318) was added in excess to inverted HLA-DR1/CLIP106–120 or canonically bound HLA-DR1/ CLIP102–120 in the presence of HLA-DM (Fig. 5B). Exchange was rapid for both samples, and the resultant spectra were fully superimposable. Therefore, as illustrated in the scheme in Fig. S6A, HLA-DM catalyzes both flipping of CLIP and, independent of the CLIP orientation, exchange against HA. Discussion Although an imposing number of peptide–MHC complexes have been successfully crystallized for both MHC classes, investigation of MHCs in solution remains a great challenge. Some information could be obtained on MHCII complexes by detecting the bound peptide (17, 18). NMR analysis of the MHC protein itself was only possible for the MHCI-β2-microglobulin (19–21), a murine single-chain construct of an MHCI heavy chain fragment (22), and a fragment of a murine class II MHC (23). In this study we use backbone NMR assignment of an MHCII subunit to investigate the dynamic reorientation of CLIP variants in complex with HLA-DR1. Complementary, the crystal structures of three HLA-DR1/CLIP complexes delineate the structural requirements for bidirectional binding. They show that the N-terminally shortened CLIP106–120 variant in its canonical orientation lacks hydrogen bonds formed by the P−2 residue in the longer CLIP102–120 peptide. Peptide inversion of CLIP106–120 reinstalls these hydrogen bonds and likely drives reorientation. CLIP106–120 shows complete realignment, and even the longer CLIP102–120 still leads to ≈50% inversion. In this case, the N-terminal hydrogen bonds can be formed in both orientations. Hence, hydrogen bonding and pocket accommodation for residues P1 to P9 is equally favorable in both alignments. Our observations show that there are no general restrictions for inverted binding of peptides to MHCII. The pseudosymmetry of CLIP might facilitate a bidirectional binding mode, but it is unlikely that the promiscuous binding pockets of MHCII are incompatible with the flipped orientation in any of the thousands of peptides that are amenable to MHCII loading within the cell. Although inversely bound peptides might be the exception, we have to note that roughly half of the crystallized unique MHCII– peptide complexes were obtained from single-chain constructs where the peptide is tethered to the N terminus of the β-chain, which is incompatible with a reversed orientation. The observation that MHCII epitope prediction algorithms, which assume fi Fig. 4. Spin-labeled CLIP106–120 proves the inverted orientation in solu- a canonical binding mode, are currently signi cantly less reliable tion. (A) Scheme of the primary sequence of CLIP106–120 carryinganN- than those for MHCI (24, 25) could indicate that in addition to terminal TEMPO moiety as site-specific paramagnetic relaxation enhancer. different binding registers a fraction of the respective MHC (B)CLIP106–120 carryinganN-terminalspinlabelorwild-typeCLIP106–120 was peptidomes are complexed in the noncanonical binding mode. loaded to empty HLA-DR1 (only β-chain labeled to decrease complexity). 1 15 Thereby the diversity of antigens presented by MHCII could be Superimposed are H- N HSQC-NMR spectra showing HLA-DR1 loaded with further enhanced. Although the open binding cleft of MHCII wild-type CLIP106–120 (blue) and TEMPO-CLIP106–120 (red). Several resonances display significantly reduced peak intensities. (C) Intensity ratios of all un- ambiguously assigned resonances from both 1H-15N-HSQC spectra were plotted to the HLA-DRβ sequence. P indicates a proline invisible in the HSQC Spin-label–affected β-chain residues 39, 47, 49, 54–68, and 75 are marked

spectrum, and asterisks mark missing assignments. (D) Epitope mapping of in red in the crystal structure of HLA-DR1/CLIP106–120 (peptide not included residues that show signiﬁcantly reduced HSQC-peak intensities according to C. for clarity).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1014708107 Günther et al. Downloaded by guest on September 25, 2021 Fig. 5. HLA-DM catalyzes CLIP interconversion and peptide exchange. (A) Superimposed regions of 1H-15N heteronuclear single quantum coherence (HSQC) 14 15 spectra showing HLA-DR1( Nα/ Nβ)/CLIP102–120 when freshly co-refolded (blue) or at indicated time points (red). Representative resonances indicate an increasing population of inverted CLIP102–120 over time. Rapid interconversion of the same resonances is seen when equimolar amounts of HLA-DM were 1 15 14 15 added before spectral acquisition (Right). (B) Superimposed H- N-HSQC spectra showing HLA-DR1( Nα/ Nβ)/CLIP102–120 representing canonical CLIP (blue), HLA-DR1/CLIP106–120 representing inverted CLIP (red), and spectra of these two samples after HLA-DM–catalyzed exchange against HA306–318 (light and dark gray, respectively). The latter two spectra superimpose well, indicating the formation of HLA-DR1/HA306–318 from both HLA-DR1/CLIP complexes.

molecules allows for orientational promiscuity, we do not expect foreign and could contribute to the antigenic component of au- fi MHCI molecules to show the same behavior. For MHCI com- toimmune phenomena. As a rst step toward such a model, future IMMUNOLOGY plexes the charged termini of the peptide are counterbalanced by research has to show that inverted MHCII–peptide complexes charged MHC residues, thereby strongly favoring peptide bind- reach the surface of the cell and thereby invoke physiologically ing in a canonical orientation. relevant T-cell responses. In the cell the preference for one CLIP orientation should be modulated by anchoring residues as well as the presence of the Materials and Methods fi P−2 residue, which is influenced by the action of proteases in Expression and Puri cation of Proteins. Recombinant bacterial HLA-DR1 the endolysosomal compartment (26). Additionally, formation of was produced and refolded in milligram amounts as described previously the thermodynamically stable complex will depend on the activity (15, 33). HLA-DM was produced in stably transfected Schneider cells as fi and hence the cell type-specific regulation of HLA-DM. For ex- previously described (2). Details of the puri cation protocol can be found in SI Materials and Methods. ample, in a B lymphoblastoid cell line deficient for HLA-DM, – CLIP106 120 was the most prominent invariant chain fragment Crystallization of HLA-DR1 with Variants of CLIP. All HLA-DR1/CLIP samples presented by HLA-DR1 (27). Moreover, a short CLIP106–121 were either produced by co-refolding HLA-DR1 and peptide or by posterior variant in complex with HLA-DR1 could be detected by mass loading with subsequent gel filtration. Samples were concentrated to 10 mg/ spectrometry at the surface of dendritic cells upon maturation, mL before being subjected to crystallization. X-ray diffraction data were coinciding with the down-regulation of HLA-DM (3). In these collected at 100 K on beamline BL14.1 at the BESSY synchrotron (Berlin, cases the formation and surface preservation of the kinetically Germany). Additional information is available in SI Materials and Methods. trapped canonically bound CLIP is probable, whereas antigen- presenting cells with high HLA-DM activity should favor the High-Field NMR Spectroscopy of MHCII Molecules. NMR spectra were acquired formation of inverted MHCII-CLIP. These complexes are likely on Bruker AV700 MHz or 900 MHz Avance spectrometers equipped with triple-resonance cryoprobes. To decrease spectral complexity only the DRβ to be presented on the cell surface, particularly if no higher- 15 fi subunit was labeled with N. For assignment of the DRβ backbone reso- af nity antigen is available for MHC alleles forming stable nances, we acquired standard 3D experiments with 15N-13C-2H–labeled DRβ CLIP complexes (as for example HLA-DR1). Assuming that— 2 α fi in complex with H-labeled DR in the presence of the respective peptides. depending on MHC/CLIP af nity, protease, and HLA-DM More details about the acquired NMR spectra and assignment strategy are activity—CLIP bound in both orientations reach the cell surface, given in SI Materials and Methods. what would be the immunological consequence? The generalized structural hallmarks for the formation of the trimeric MHC– Peptides, Loading of MHC, and Peptide Exchange by HLA-DM. Peptides were peptide–T cell receptor (TCR) complex indicate the interactions synthesized using Fmoc-based solid phase chemistry. They were used for for the encountering TCR to center around the middle of the loading in 10-fold molar excess during protein refolding and 10- to 20-fold MHC-bound peptide (28, 29). Although a certain variability in molar excess for loading a posteriori in the presence of the dipeptidic MHC this docking mode has been observed for autoimmunogenic loading enhancer Ac-FR-NH2 to accelerate the reaction (34). To exchange – – CLIP106–120 or CLIP102–120 for HA306–318, HLA-DM was added to isolated HLA- peptide MHC complexes (30 32), the surface charge reversal of DR1/CLIP complexes in stoichiometric amounts, followed by addition of HA. the two CLIP orientational variants (Fig. S6B) would likely en- CLIP numbering refers to the amino acid sequence of the longer p35 human gage different TCRs. Moreover, assuming that differential N- CLIP variant, as described previously (35). In regard to the shorter p33 iso- terminal processing occurs for self-peptides other than CLIP, it form, our peptides refer to sequences 90–104 and 86–104, respectively. will be of interest to probe likely candidate peptides for bidirectional binding to MHCII alleles. Such a finding would be ACKNOWLEDGMENTS. We thank Michael Beyermann for standard peptide of biological relevance if there is an orientational difference be- synthesis; Peter Schmieder for the setup of certain NMR pulse sequences; Ronald Kühne for helpful discussions; and Miriam-Rose Ash for critically tween presentation of a particular self-peptide in the thymus reading the manuscript. C.F. was supported by grants from the Bundesmi- during positive and negative selection of T cells and later in the nisterium für Bildung und Forschung (01 EZ 1010B) and from the Deutsche periphery. In this case, T cells could recognize the self-peptide as Forschungsgemeinschaft (SFB765).

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