HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism

Abstract

MHCII proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The non-classical MHCII protein HLA-DM chaperones peptide-free MHCII against inactivation and catalyzes peptide exchange on loaded MHCII. Another non-classical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we use x-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the alpha subunit 310 helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis demonstrates that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic and place constraints on possible functional roles for HLA-DO in antigen presentation.

The Binding of TAPBPR and Tapasin to MHC Class I Is Mutually Exclusive

“…It was shown recently that HLA-DO is a MHC class II mimic and binds tightly to HLA-DM, directly suppressing HLA-DM from interacting with MHC class II (39). Because there is no direct association of TAPBPR with tapasin, mechanistically TAPBPR does not appear to work in the same manner as HLA-DO.…”

Susceptibility to HLA-DM Protein Is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide

“…The structure was solved by molecular replacement with six copies per asymmetric unit using the known structure of DR1-A2(104 -117) (31). Data processing, model building, and refinement were performed as described (30). Data processing and refinement statistics for the final model (PDB code 4OV5) are shown in Table 2.…”

“…Bio-layer interferometry experiments of DM binding to immobilized DO were carried out on a ForteBio Octet instrument (Pall Life Sciences) as described previously (30).…”

“…DM bound to DR1 carrying a covalently bound, N-terminally truncated peptide shows extensive rearrangement of the MHCII pocket 1 (24). DM bound to DO, a natural inhibitor that acts as a DM substrate mimic, shows similar rearrangements in the same region (30). Both structures were interpreted as representing a transient intermediate in the DM-MHCII-peptide exchange reaction, which would resolve upon peptide binding and displacement of DM.…”

“…In each case we substituted DR1 residues for those found in DO, a structural homolog that acts as a tight-binding competitive inhibitor (30). We constructed three mutants: H33A,A37K in the strand s3-s4 loop, a region that changes conformation in the DM complex (24,30); P16Y,Q18K located at similar position but in the strand s1-s2 loop as a control; and T41A in the s4 strand where it contacts the DR1 extended strand region ␣51-57 implicated in DM modulation of DR1-peptide hydrogen-bonding interaction (17,23). Both H33A,A37K and T41A mutations had dramatic effects in increasing DM binding, whereas the P16Y,Q18K mutation had no effect (Fig.…”