Structural basis for the association of the HLA-DRB1 locus, citrullination and rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting approximately 0.5-1% of the Western population. The disease is characterized by synovial joint inflammation, joint pannus and bone erosion, and leads to comorbidities such as lung fibrosis and atherosclerosis, reducing the expected life span of patients by up to 10 years. The human leukocyte antigen (HLA) locus plays a vital role in immunity, encoding highly polymorphic molecules that present peptides to T cell lymphocytes. Whilst the pathogenesis of RA is still unclear, genetic studies have shown that it is associated with the HLA-DRB1 locus. Specifically, the association corresponds to amino acids 70 to 74 in the third hypervariable region of the HLA-DRB1 allele, commonly referred to as the ‘shared epitope’ (SE). These amino acids line the fourth anchoring pocket (P4) of the HLA-DRB1 molecule and therefore have the ability to discriminate which peptides are capable of being presented. In SE allomorphs, the P4 pocket has an overall positive charge, consequently disfavouring positively charged sidechains from occupying this pocket in preference for negatively charged, polar or hydrophobic residues. Interestingly, the SE is associated with the presence of anti-citrullinated protein antibodies (ACPA) in RA patients. Citrullination is the conversion of the positively charged arginine to the polar or neutral citrulline. Previous studies have shown that citrullination of self-antigens can significantly increase the affinity of peptides for SE allomorphs. It is hypothesized that citrullination of peptides in the inflamed joints of RA patients allows these peptides to bind to HLA-DRB1 allomorphs possessing the SE, generating neo-antigens and ultimately causing autoreactivity. However there is no structural data to describe how SE allomorphs preferentially bind to citrullinated self antigens. This thesis presents the structural basis of citrullinated vimentin and aggrecan peptide presentation by the RA susceptible, SE allomorphs, HLA-DRB1*0401 and HLADRB1*0404. Citrulline was accommodated in the positively charged P4 pocket of SE allomorphs, where Lys71β of HLA-DRB1*0401 or Arg71β of HLA-DRB1*0404, performed a key discriminatory role. In addition, Ala74β of the SE motif, provided space for the P4-Cit to adopt an upright configuration, and explains why Ala74β is conserved in all SE allomorphs. In contrast, HLA-DRB1*0402, an RA resistant allomorph that encodes a negatively charged P4 pocket, accommodated both arginine and citrulline. Together, these studies provide a structural basis for the preferential binding of citrullinated peptides to SE allomorphs. The Native American population has an increased prevalence of ACPA+ RA compared to other ethnic groups, and is likely attributable to the high frequency of the SE allele HLADRB1*1402 in this population. HLA-DRB1*1402 exhibits the same SE motif as HLADBR1*0404, and thus may prefer to bind citrulline to arginine in its electropositive P4 pocket. However, HLA-DRB1*1402 also possesses a Ser11β and Ser13β, residues thought to confer resistance to RA. Therefore, we determined the crystal structure of HLADRB1*1402 in complex with native and citrullinated vimentin peptides. Interestingly, the electropositive P4 pocket was able to accommodate both an arginine and a citrulline. However, these residues adopted starkly different conformations, with the arginine buried deep within the P4 pocket, away from the electropositive Arg71β, and instead interacted with Ser11β and Ser13β. In contrast, the P4 citrulline adopted an upright constrained U-type conformation, and formed a hydrogen bond with Arg71β. This result suggests that T cell receptors on T cells could discriminate between the differing configurations of the native and citrullinated peptides. Collectively, these findings provide a molecular basis for the association of HLA Class II SE allomorphs and citrullination in RA pathogenesis. More broadly, this thesis highlights the important role of post-translational modifications in autoimmune disease, and will aid the development of vaccine-based therapies.