Crystal structure of HLA-DP2 and implications for chronic beryllium disease

Abstract

Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.

Fig. 1.

Overview of the DP2-pDRA structure. (A) Ribbon representations of the full DP2 α (cyan) and β (magenta) extracellular domains and a wireframe representation of the pDRA peptide with Corey–Pauling–Koltun (CPK) coloring viewed from the β-chain side. (B) Top view as in A, but without the α2 and β2 domains. Individual amino acids in the peptide are labeled.

Fig. 2.

Unusual properties of the DP2 peptide-binding groove. (A) Details of the p1 (first panel), p4 (second panel), p6 (third panel), and p9 (last panel) binding pockets are shown. In each panel, the semitransparent water-accessible surfaces of the DP2 α1 (cyan) and β1 (magenta) domains are shown, as well as wireframe representations of the side chain of the pDRA amino acid at that position (CPK coloring) and the DP2 amino acids lining the pocket (CPK coloring, except for carbons: DP2 α1 carbons, dark cyan; and DP2 β1 carbons, dark magenta). (B) Three MHCII structures were overlaid on the basis of their α1 domains: DP2-pDRA (cyan), HLA-DR3 bound to the invariant chain CLIP peptide (PDB ID code 1A6A, orange) and mouse IA^b bound to CLIP peptide (PDB ID code 1MUJ, magenta). (Left) MHCII α-helices as ribbons and the peptide from p1 to p9 as a CA stick. (Right) Full β1 domains as ribbons. (C) Three MHCII structure were overlaid on the basis of the four central β-strands of the floor of the peptide-binding groove: DP2-pDRA (cyan), mouse IE^k bound to a peptide from pigeon cytochrome c (PCC; PDB ID code 1KTD, orange) and mouse IA^k bound to a peptide from hen egg lysozyme (HEL; PDB ID code 1IAK). The figure shows the four central β-strands as ribbons, the peptide from p3 to p6 as a CA stick, and a wireframe representation of the side chain of the p4Leu in each structure.

Fig. 3.

βGlu69 lies in a solvent-exposed acidic pocket. (A) The water-accessible surface of the DP2 molecule (without bound pDRA) is shown in the area between p5Pro and the DP2 β-chain α-helix colored by the relative charge of the surface atoms (red, negative; blue, positive) (Swiss PdbViewer). A wireframe representation of pDRA is also shown with CPK coloring. (B) Same view as A but with ribbon representations of DP2 α (cyan), DP2 β (magenta), and pDRA (yellow). Also shown are wireframe representations of the side chains of βGlu26, βGlu68, and βGlu69 with CPK coloring. (C) View of the acidic pocket looking down the peptide-binding groove from the C terminus of pDRA. Portions of the DP2 β-chain α-helix and β-sheet are shown as magenta ribbons. Wireframe representations of the side chains of βGlu26, βTyr28, βGlu68, and βGlu69 are shown with magenta carbon and red oxygen. A wireframe representation of p4 to p6 of pDRA is shown with yellow carbon, red oxygen, and blue nitrogen. Also shown is a wireframe representation (CPK coloring) of the side chain of the pDRA p-2Arg from a neighboring symmetry-related DP2-pDRA molecule. Green lines connect oxygens in the acidic pocket that potentially could form H bonds or salt bridges to each other or to the nitrogens of the arginine guanidinium group. (D) The same view as in C. In this case, the 2Fo-Fc electron density map around βGlu26, βTry28, βGlu68, and βGlu69 and p-2 Arg is shown, contoured to 1σ.

Fig. 4.

DP2 βGlu26, βGlu68, and βGlu69 are essential for T-cell recognition of Be. (A) Staining of DAP.3 L cells for surface expression of HLA-DP2. DAP.3 L cells transfected to express either WT DP2 or a DP2 mutant (DP2-β26E > A, DP2-β26E > Q, DP2-β68E > A, and DP2-β69E > K) were stained with either an IgG1 isotype control (open) or an anti-HLA-DP-specific mAb, B7.21 (filled). (B) Intracellular IFN-γ expression in a Be-responsive T-cell line in response to BeSO₄ or SEB presentation by fibroblasts expressing either WT DP2 or a DP2 mutant is shown. The percentage of CD4⁺ T cells expressing intracellular IFN-γ is shown in the upper right quadrant of each density plot. (C) T-cell proliferation of the same T-cell line using BeSO₄-pulsed mitomycin C-treated fibroblasts expressing either WT DP2 molecule or DP2 mutants is shown. The data are expressed as the mean cpm ± SEM.