Crystal structure of the HLA-DM-HLA-DR1 complex defines mechanisms for rapid peptide selection

Abstract

HLA-DR molecules bind microbial peptides in an endosomal compartment and present them on the cell surface for CD4 T cell surveillance. HLA-DM plays a critical role in the endosomal peptide selection process. The structure of the HLA-DM-HLA-DR complex shows major rearrangements of the HLA-DR peptide-binding groove. Flipping of a tryptophan away from the HLA-DR1 P1 pocket enables major conformational changes that position hydrophobic HLA-DR residues into the P1 pocket. These conformational changes accelerate peptide dissociation and stabilize the empty HLA-DR peptide-binding groove. Initially, incoming peptides have access to only part of the HLA-DR groove and need to compete with HLA-DR residues for access to the P2 site and the hydrophobic P1 pocket. This energetic barrier creates a rapid and stringent selection process for the highest-affinity binders. Insertion of peptide residues into the P2 and P1 sites reverses the conformational changes, terminating selection through DM dissociation.

Figure 1. Overview of the HLA-DM – HLA-DR1 complex

(A) Generation of a covalent DM-DR1 complex with a linked peptide that lacks three N-terminal residues. (B, E) Top-view of the DM-DR1 complex shows DM interaction site on the DRα chain and a partially empty peptide binding groove. DMα is colored red, DMβ yellow, DRα cyan and DRβ blue. Peptide and the DR αV65C covalent attachment site are colored orange. (C, D, F) Side views of the DM-DR1 complex, with contact residues of DR1 (C) or DM (D) presented as surfaces, emphasizing interactions between DRα and DMα chains. The C-termini of DRβ and DMβ chains are indicated (C). (F) The location of the peptide N-terminus in the DR1-HA_306–318 structure is indicated with an arrow. See also Figure S1 as well as Movies S1 and S2.

Figure 2. Key residues involved in the DM – DR1 interaction

(A) Molecules are shown in cartoon representation, and interacting residues are surface rendered. Mutations that affect the interaction are indicated. (B) List of DM and DR mutants (new and previously identified), grouped by functional activity (new mutations are underlined). These residues are located at the DM-DR1 interface, except DR αP96 (mutation introduces glycan), DM βA55 (residue not surface exposed) and DM βE8 (mutation to lysine may affect DM conformation). DR αF51 and αE55 have moved into the peptide binding groove in the DM-DR1 complex, as described in Figures 4 and 5. (C) Conservation of key contact residues. Alignments focus on the DR1 α1 domain (res. 31–61) and two segments of DMα (res. 91–101 and 121–141). Interacting residues are colored blue (DRα) and yellow (DMα). Sequences were retrieved from the www.ensembl.org website (June 2012). Key DRα - DMα contact residues are highlighted in red; DR residues that have move into the groove are colored green. See also Figure S2.

Figure 3. HLA-DR αW43 is critical for HLA-DM binding

(A) Structure of DR1-HA_306–318 complex showing packing interactions of DR αW43 (red) with hydrophobic residues, including peptide P1 tyrosine (green). (B) W43 stabilizes P1 pocket in DR1-HA_306–318 (white) but is rotated away from the P1 pocket in the DM-DR1 complex (red). Conformational changes in DM-DR1 (cyan) compared to DR1-HA_306–318 (yellow) include movement of β strands and extension of the helix to include DR α52–55. (C) Overall conformation of DR binding groove in DM-DR1 complex compared to DR1-HA_306–318. (D) Hydrogen bonding network established by DR αW43 and αK38/αE40. (E) Pocket on DM surface occupied by DR αW43. (F) Effect of DM αN125 mutations on rate of peptide binding, measured using a real-time peptide binding assay based on fluorescence polarization (FP). Binding of a fluorescent peptide to a large protein reduces its tumbling rate in solution (measured in mP units). Graph shows change in rate of initial peptide binding (mP min⁻¹). See also Figure S3.

Figure 4. Large conformational changes stabilize the empty binding groove around the P1 pocket in the DM-DR1 complex

(A) Surface representation of DR1 with fully bound HA_306–318 peptide showing αF51 (yellow), αF54 (green) and αE55 and βN82 (both red). αF51 is pointing out of the groove and αE55 is located outside the binding site. DR βH81 (dotted line) is rendered transparent to improve visibility of DR βN82. DR β82 forms bidentate hydrogen bonds with the peptide backbone at P2. (B) In the DM-DR1 complex, αF51 has moved into the P1 pocket, stabilizing this hydrophobic site. DR αE55 has moved into the groove where it forms a water-mediated hydrogen bond with DR βN82. See also Figure S4.

Figure 5. Stabilization of P1 site by DR αF51 and DR βF89

(A) Top view of DR1-HA_306–318 complex, with peptide P1 tyrosine (green) in P1 pocket. (B) DR αF51 moved into highly hydrophobic P1 pocket in DM-DR1 complex. (C&D) DR βF89 (red) also moved into groove in DM-DR1 complex, stabilizing αF51. Position of DR βF89 (*) in DR1-HA_306–318 complex is indicated in grey (C). (E) SPR analysis of DM binding by DR αF51 mutants: injection of DR-CLIP (stage 1, DR binding), buffer (stage 2, dissociation of empty DR), peptide (stage 3, peptide-dependent dissociation of DM-DR complex). (F) Kinetics of peptide binding by DR αF51 mutants +/− DM. Peptide binding was examined in a real-time fluorescence polarization (FP) readout (mP units), as explained in Figure 3F. See also Figure S5.

Figure 6. Conformational differences between pH 6.5 and pH 5.5 DM-DR1 structures

(A & B) Peptide electron density at pH 5.5 (A) and pH 6.5 (B). (C) Differences in position of key DR residues at pH 6.5 (colored) and pH 5.5 (gray); pH 5.5 residues (*). (D) Space filling model of hydrophobic DR residues of P1 pocket in pH 6.5 structure, DR αF51 (yellow) located partially in P1 site and βF89 (red) outside the groove. (E & F) Differences in the interaction of an acidic pair (DMβ D31-E47) with DR1 at pH 5.5 (E) and pH 6.5 (F). See also Figure S6.

Figure 7. Model for key steps in DM – DR interaction

(1) CLIP is bound in DR groove and DR αW43 (red, arrow) stabilizes P1 pocket. (2) Peptide N-terminus dissociates from DR groove and DR αW43 rotates away from the P1 pocket, becoming available for interaction with DM. Other DR residues (arrows) move into groove during transition to DM-bound state. (3) DM stabilizes empty DR, and DR αF51 and βF89 protect the hydrophobic P1 pocket. (4) Rapid binding of peptides to partially accessible groove; peptides that do not successfully compete with DR residues (yellow) for P2 site and P1 pocket are not stably bound. (5) Binding of the peptide N-terminus reverses conformational changes and results in DM dissociation.