HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

Abstract

BackgroundHLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive.Methodology/Principal FindingsWe have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties.Conclusion/SignificanceThis study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a “compare-exchange” sorting algorithm on an available peptide pool. Such a “third party”-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

Cutting Edge: HLA-DM–Mediated Peptide Exchange Functions Normally on MHC Class II–Peptide Complexes That Have Been Weakened by Elimination of a Conserved Hydrogen Bond

“…Therefore, we have investigated the effect of DM on the folding-unfolding of the complex and how this may be related to the mechanism underlying the peptide exchange reaction. We have shown: 1) the requirement of DM for an exchange peptide at equimolar or greater concentration than the preformed complex to promote prebound peptide release; 2) the absence of measurable cooperativity in the release of the prebound peptide, probably due to a simultaneous disruption of the interactions between MHC II and peptide mediated by DM; and 3) the exchange ligand needs to fold into the groove more efficiently than the prebound peptide to displace it (6).…”

“…Peptides derived from the sequence of wt hemagglutinin (HA) 306-318 (G) PKYVKQNTLKLAT have been synthesized as described previously (3,4,6) and are listed in Supplemental Table I and II.…”

“…Dissociation of peptides from DR1 and b81mut was measured with fluorescence polarization (FP) spectroscopy essentially as described (6). The t 1/2 values of the various complexes are reported in Supplemental Table I and II.…”

“…Peptide K D values for DR1 and b81mut were assessed in equilibrium-based competition binding assay as previously described (6). Affinity values are reported in Supplemental Table I.…”

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

“…Because the immunogenicity of epitopes after infection and vaccination is strongly linked to their relative DM susceptibility (8,13,58,59), understanding these determinants is crucial to follow immune responses, improve vaccines, and understand the etiology of autoimmune disease. Previous models for predicting DM susceptibility have variously implicated particular conserved hydrogen bonds near pocket 1 (17,18,20,21), spontaneous dissociation of the peptide N terminus (22,60), conformational lability of the 3 10 helical region adjacent to pocket 1 (23), an SDS-sensitive flexible conformation determined by pocket 1 occupancy (28,45), and an "compare-exchange-pushoff" mechanism (25). Although these different approaches have generally implicated interactions around the pocket 1 region (61), some studies are consistent with more distributed effects (16,25).…”