HLA–DM Senses Peptide‐MHC Class II Interactions Throughout the Peptide Binding Groove
Authors: Reyes‐Vargas Eduardo , Barker Adam P , Zhou Zemin , He Xiao , Jensen Peter E 
Published: November 2023

The major histocompatibility complex class II (MHCII) antigen‐processing pathway is critical for adaptive immunity as the mechanism through which ligands are generated for recognition by CD4 T‐cells. Within the pathway, human leukocyte antigen DM (HLA‐DM) edits the repertoire of peptides that are presented to CD4 T‐cells by catalyzing multiple rounds of peptide exchange and accelerating peptide loading on MHCII molecules. In endosomes, HLA‐DM first catalyzes the dissociation and exchange of the proteolytically processed Ii derived Class II‐associated invariant chain peptide (CLIP) bound to MHCII molecules through a mechanism that follows Michaelis‐Menten kinetics and involves a transient interaction with the peptide‐MHCII complex. HLA‐DM then continues to catalyze the rate of dissociation and exchange of all other peptides. The catalytic potency of DM varies such that peptide exchange is markedly enhanced on some peptide‐MHCII complexes and not others. This capacity to differentially edit peptide‐MHCII complexes has important biological implications through its effect of skewing the repertoire of foreign and self‐peptide complexes available for activation or tolerance induction of CD4 T‐cells. Despite the large body of evidence indicating the central role of HLA‐DM in the MHCII antigen‐processing pathway, the catalytic mechanism and the rules that govern the susceptibility of peptide‐MHCII complexes to DM mediated editing are not completely understood. Evidence suggests that unstable interactions at the N‐terminus of bound peptides is the key determinant of peptide‐MHCII complex sensitivity to HLA‐DM and that partial dissociation of a peptides N‐terminus is necessary for this sensitivity. Herein, we employed a fluorescence polarization assay to independently assess the effective affinity and catalytic turnover, components of the catalytic mechanism, in real‐time kinetic measurements of DM activity. Using a series of amino acid substituted peptides, we have analyzed the effects of disfavored interactions on the kinetics of DM editing. We find that disruptions throughout the peptide binding groove contribute to the effective binding affinity of DM for a given peptide complex and to the rate of peptide dissociation from the DM–peptide‐MHCII catalytic complex. Experiments with mutant peptide‐MHCII complexes that disrupt elements in the conserved hydrogen bond network show that disruption near the C‐terminus results in increased DM affinity and catalytic turnover. Furthermore, we found that complexes with an N‐terminal truncated form of the immunodominate HA peptide, resulting in the loss of three H‐bonds, showed an unexpected increase in catalytic turnover when compared to the unmodified HA‐MHCII complex. This result was even more pronounced when a conserved bidentate H‐bond forming residue near the P1 anchor of the HA‐MHCII complex was disrupted by site‐directed mutagenesis. Together, this implies that peptide‐MHCII interactions near the N‐terminal segment of a peptide are intact during the functionally active DM‐peptide‐MHCII catalytic complex and that interactions at the DM‐MHCII contact interface are intimately linked to unstable interactions throughout the peptide‐binding groove.Support or Funding InformationSupported in part by NIH grants AI30554 and AI33614