Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection

Abstract

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.

Fig. 1. dsA2 is stable at very low dipeptide concentrations.

Thermal denaturation of A2 in the presence of increasing concentrations of GM measured by nanoDSF (tryptophan fluorescence). a, b First derivatives of the F350/F330 (ratio of fluorescence emissions at 350 and 330 330 nm) curves for wtA2 a and dsA2 b with the maxima reporting the melting temperatures (T_m). The concentration of GM (in mm) is indicated in rainbow colors from red (maximum, 100 mm) to blue (lowest, 0.05 mm). The arrow points to the transition of the dsA2 heavy chain that is visible even at very low dipeptide concentration. c Scatter plot of the T_m obtained for the A2 heavy chain (first unfolding event in A) as a function of GM concentration (mmΜsmall M).

Fig. 2. Stability and peptide binding of dsA2 measured by native MS.

a Representative mass spectra of dsA2 and wtA2, 10 µm, respectively, in 250 mm ammonium acetate, pH 8 recorded at a collision energy of 50 V. Pink, β₂m; green, A2 heavy chain (A2-HC); yellow, A2 complex. Comparing the spectrum of dsA2 alone (top) with the spectrum of dsA2 in the presence of 0.5 mm GM (second panel), the low m/z range (zoom) clearly shows that the protein complex no longer contains any dipeptide used for refolding. Note, the dipeptide owing to low-affinity dissociates easily at the activation energies necessary to resolve the peaks sufficiently. The stability of dsA2 does not depend on the presence of dipeptide (top), whereas wtA2 without GM is hardly visible (third panel). In presence of 0.5 mm GM, the wtA2 complex can be detected in high abundance, although the wtA2/GM complex is not observed (bottom). b Superstoichiometric binding of NV9 high-affinity peptide to dsA2. Green, dsA2 heavy chain; yellow, dsA2 complex; blue, dsA2/NV9 complex. Mass spectrum of dsA2 at collision voltage of 50 V is given as a reference (top). At low collision voltage (10 V), the peptide-bound complex is the most-abundant species upon addition of 0.1 mm NV9 (middle). When increasing the voltage to 50 V, the complex begins to dissociate. However, some heavy chain/NV9 peptide complex can be detected (8+ peak), indicating high stability (bottom). c Substoichiometric binding of NV9 high-affinity peptide to dsA2. Pink, β₂m; green, dsA2 heavy chain; yellow, dsA2 complex; blue, dsA2/NV9 complex. Reference mass spectrum of dsA2 at 50 V collisional energy (top). Peaks for dsA2/NV9 are gathered from a mixture of dsA2 and high-affinity nonapeptide NV9 (10:1), whereas the low-affinity control YF9 gives no signal (second panel). The 12+ peak at 3723 m/z was selected for MS/MS analysis at 10 V in the collision cell (third panel). The MS/MS spectrum measured at 100 V clearly shows the dissociation of the complex into dsA2 as well as its subunits and NV9 (bottom).

Fig. 3. Crystal structures of dsA2 in complex with dipeptides in the A and F pockets.

a Overlay of the peptide-binding groove with a ribbon diagram of dsA2/GL in gold and wtA2/GLCPLVAML in gray. The C84-C139 disulfide bond is shown as sticks. b The dsA2/GM₂ structure (gold; see Supplementary Table 2) is shown overlaid with the published wtA2/GLCPLVAML structure (pdb 3MRF; gray). View from the side of the α₂ helix with comparison of the GLCPLVAML peptide (gray) with the GM dipeptides (cyan) in the A and F pocket (peptides depicted as sticks). A transparent cartoon of the secondary structures of the peptide-binding groove is shown for orientation. c, d Zoom into the F pocket of the same structure, showing that the carboxylate group of the GM dipeptide is in the same orientation as the carboxyl group of the full-length peptide, despite the Tyr84Cys mutation. In dsA2/GM₂, water molecules (W1, W2, W3, and W4) fill the space where the side chain of Tyr84 is pointing in wtA2/GLCPLVAML.

Fig. 4. The locked and unlocked states of the A and F pockets of A2.

a Ribbon diagram of the dsA2/peptide_free-1 crystal structure, showing electron density from a 2mF_o–DF_c map for the water molecules (gray) and the EDO molecules (green) in the peptide-binding groove at a contour level of 1σ. b Close-up of the A pocket in the locked (cyan, from the dsA2/GM structure) and the unlocked (gold, from the peptide-free structure) states. In the unlocked state, the hydrogen bond between Tyr99 and His70 is broken and the side chains of His70 and Phe9 move into the peptide-binding groove, whereas the side chain of Tyr99 moves downwards. c Systematic analysis of the distance distribution between the OH atom on Tyr99 and the closest nitrogen atom on the imidazole side chain of His70 for all peptide-loaded HLA-A0201 molecules observed in the PDB. The histogram shows that >90% of the deposited structures contain a hydrogen bond between His70 and Tyr99. d, e Two alternate hydrogen bond networks that are formed as a result of the presence of the methionine side chain in the F pocket are seen in each dsA2/GM₂ molecule. This leads to conformational changes in the side chains of residues His74 and Phe9.

Fig. 5. Superposition of the unlocked and locked states of the F pocket and comparison of the electrostatic surface of the peptide-free and -occupied dsA2 molecules.

a The unlocked state (derived from the dsA2/peptide_free-1 structure) is in gold, and the locked state (from the dsA2/NLVPMVATV structure) in cyan. The side chains involved in locking the F pocket are His74, Asp77, Arg97, His114, and Tyr116. The adjacent residues involved in locking the A pocket (Phe9, His70, and Tyr99) are also depicted to show their close proximity. b Top view of the F pocket in the unlocked state showing that the conformations of the side chains of the peptide-free dsA2 molecule coincide with the side chain conformations of one of the conformational states of dsA2/GM₂ structure, where the methionine side chain is not involved in interactions with the side chains lining the F pocket. The only difference is a hydrogen bond between His74 and Asp77 in the peptide-free dsA2 structure (yellow dotted line). c Electro-surface potential of the peptide-binding groove of peptide-loaded dsA2 (+NV9), dsA2/GM₂ (+GM), and peptide-free dsA2 (empty). The A and F pockets are marked to show their opening up, and the accompanying charge shifts, in the unlocked state.

Fig. 6. The dynamics of the transition between the unlocked and the locked states of the F pocket demonstrated by molecular dynamics simulations.

a The graphs show the calculated free energy along the Tyr116 χ1 dihedral angle coordinate obtained from umbrella sampling free energy simulations (black: empty dsA2, red: dsA2/NV9, green: dsA2/GM₂). The three structure images correspond to the states along the dihedral reaction coordinate indicated by the arrows: left, Tyr116 χ1 in near-trans (i.e., locked) conformation as found in the peptide-bound state; center, Tyr116 χ1 in an energetically unfavorable transition state; right: Tyr116 χ1 in -gauche close to the unlocked conformation found in the peptide-free or GM bound states of dsA2. b Distribution of sampled side chain dihedral angles of residues near Tyr116 in the case of Tyr116 χ1 being in the trans regime (locked, peptide-bound state, black curve [blue for Arg97 χ 3]) and in the case of Tyr116 χ1 being in the -gauche regime (unlocked state, red line [green for Arg97 χ3]).