The interplay between citrullination and HLA-DRB1 polymorphism in shaping peptide binding hierarchies in rheumatoid arthritis

Abstract

The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the β-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA-DR4-self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA β-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide-HLA-DR4 binding affinities in RA.

Keywords: X-ray crystallography; arthritis; major histocompatibility complex (MHC); mass spectrometry (MS); structural biology.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

Figure 1.

Motif analysis of naturally presented peptides. DRB1*04:05 peptide repertoire analysis reveals amino acids that are more (positive values) or less (negative values) represented in the DRB1*04:05 9-mer cores (n = 659) compared with frequencies found in human proteome (A). Heat map depicting position-specific (columns 1–9) amino acid (rows) differences in 9-mer cores of the DRB1*04:05 compared with *04:01 and *04:04 on a scale of −100% (decreased in DRB1*04:05) to 100% (increased in DRB1*04:05) (B). A marked increase in Asp (21%), and Glu (32%) at P9 was observed in DRB1*04:05 (n = 659) compared with either *04:01 (n = 241) or *04:04 (n = 265).

Figure 2.

Titration curve of self-peptide binding reflected by displacement of reporter fluorescent peptide in HLA-DRB1*04:01 (A), HLA-DRB1*04:04 (B), and HLA-DRB1:04:05 (C). Each data point represents normalized relative binding (in percentage) from three independent experiments. Mean values are plotted, and error bar showed SE Nat, native peptide without citrulline; Cit, citrullinated peptide. Curve fit for each set of peptides for all three allomorphs HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1:04:05 are displayed in Table S2.

Figure 2.

Titration curve of self-peptide binding reflected by displacement of reporter fluorescent peptide in HLA-DRB1*04:01 (A), HLA-DRB1*04:04 (B), and HLA-DRB1:04:05 (C). Each data point represents normalized relative binding (in percentage) from three independent experiments. Mean values are plotted, and error bar showed SE Nat, native peptide without citrulline; Cit, citrullinated peptide. Curve fit for each set of peptides for all three allomorphs HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1:04:05 are displayed in Table S2.

Figure 2.

Titration curve of self-peptide binding reflected by displacement of reporter fluorescent peptide in HLA-DRB1*04:01 (A), HLA-DRB1*04:04 (B), and HLA-DRB1:04:05 (C). Each data point represents normalized relative binding (in percentage) from three independent experiments. Mean values are plotted, and error bar showed SE Nat, native peptide without citrulline; Cit, citrullinated peptide. Curve fit for each set of peptides for all three allomorphs HLA-DRB1*04:01, HLA-DRB1*04:04, and HLA-DRB1:04:05 are displayed in Table S2.

Figure 3.

Polymorphic residues in the peptide-binding cleft of HLA-SE molecules. Polymorphic residues in the P1, P4, and the P9 pocket of HLA-DRB1*04:01, DRB1*04:04, DRB1*04:05, and the shared-epitope motif are shown (A). The G86V variation on the DRβ chain of HLA-DRB1*04:04 may obstruct binding of hydrophobic residues such as Tyr in the P1 pocket (B). The P4-Cit forms a conserved hydrogen bond with the Lys-71β/Arg-71β, a polymorphic residue in the P4 pocket between HLA-DRB1*04:01 and DRB1*04:04/*04:05 (C). The P9 pocket of HLA-DRB1*04:01 and DRB1*04:04 consists of a Asp-57β that shapes the P9 pocket of these two alleles by forming a salt bridge with the conserved Arg-76α (D). The D57S variation on the DRβ chain of HLA-DRB1*04:05 forms extensive hydrogen bonds with P9-Asp in the peptide (E). The P9 pocket of HLA-DRB1*04:05 with a Ser-57β that has a shorter side chain has been shown to have a preference for negatively charged residue in the P9 position of peptides.

Figure 4.

Crystal structures of HLA-SE in complex with RA-associated antigens. The HLA-DRB1* molecule is represented as a schematic, whereas the RA-associated peptides are shown as sticks, and the electron density maps of these peptides are shown as 2Fo − Fc maps contoured at 1σ. Both HLA-DRB1*04:01 (A) and DRB1*04:04 (B) bound to the LL37–91Cit86–98 and interact with the peptide P4-Cit in a highly conserved manner despite Lys-71β/Arg-71β polymorphism in the P4 pocket. C, Arg-71β of DRB1*04:05, like the HLA-DRB1*04:01/*04:04, forms a conserved hydrogen bond with the P4-Cit in peptide vimentin-424Cit419–431. Crystal structures of HLA-DRB1*04:01 in complex with either fibrinogen-β-74Cit69–81 (D) or fibrinogen-β-72,74Cit69–81 (E), the same epitope with additional citrulline in the P2 pocket, showed no register shift upon binding to the DR molecule. The P2-Cit of both fibrinogen-β-72,74Cit69–81 and F, collagen type II-1240Cit1237–1249 bound to HLA-DRB1*04:01 are projected outward from the peptide-binding groove and are accessible by TCRs. The P4-Asp, in collagen type II-1240Cit1237–1249, like the P4-Cit, forms a hydrogen bond with the Lys-71β in HLA-DRB1*04:01. The presence of P3-Arg (G) or P3-Cit (H) in the histone2B68–82 peptide does not change the affinity of the peptide for the HLA-DRB1*04:04 and interacts with Asn-62α. The P4-Ile in the histone2B68–82 peptide liberates the Arg-71β in DRB1*04:04 to form a hydrogen bond with P7-Glu and Asp-28β. I, overlay of the eight structures showed no significant conformational changes in the HLA molecules, and the peptide alignment is highly conserved. Citrulline in the P4 pocket is presented upward in a highly conserved manner.