Small Molecules That Enhance the Catalytic Efficiency of HLA-DM

Abstract

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. Large lateral surfaces involved in the DM:HLA-DR (DR) interaction have been defined, but the mechanism of catalysis is not understood. In this study, we describe four small molecules that accelerate DM-catalyzed peptide exchange. Mechanistic studies demonstrate that these small molecules substantially enhance the catalytic efficiency of DM, indicating that they make the transition state of the DM:DR/peptide complex energetically more favorable. These compounds fall into two functional classes: two compounds are active only in the presence of DM, and binding data for one show a direct interaction with DM. The remaining two compounds have partial activity in the absence of DM, suggesting that they may act at the interface between DM and DR/peptide. A hydrophobic ridge in the DMβ1 domain was implicated in the catalysis of peptide exchange because the activity of three of these enhancers was substantially reduced by point mutations in this area.

Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

“…FP Assay-Association of MBP-488 was monitored by FP as described previously (32). The assay was performed in black polystyrene 384-well flat bottom plates (Corning, Corning, NY) with a nonbinding surface, and the reactions were protected from external light and evaporation with an aluminum foil seal (Excel Scientific, Wrightwood, CA) when the plate was not in the reader.…”

“…The experiments make use of a human Class II MHC molecule, the expression of which predisposes its carrier to multiple sclerosis, HLA-DR2 (DRA, DRB1*1501), and a photocleavable peptide based on a T cell epitope of human myelin basic protein (MBP 85-99 ) (30,31). Rapid, early binding events were followed with a fluorescence polarization (FP) readout, in which the fraction of emitted fluorescent light that retains polarization is proportional to the amount of MHCbound fluorescent reporter peptide, because of a slower tumbling speed of the MHC-peptide complex compared with free peptide (32). The presence of DM substantially accelerated peptide binding to such empty DR molecules under all of the reaction conditions tested.…”

Forced Expression of HLA-DM at the Surface of Dendritic Cells Increases Loading of Synthetic Peptides on MHC Class II Molecules and Modulates T Cell Responses

“…More recently, DM mimics capable of catalyzing peptide exchange have been described. These simple organic chemicals or peptidic molecules operate at neutral pH and enhance T cell activation (26,27,(43)(44)(45). Interestingly, a better loading was obtained by combining n-propanol and DMY, suggesting that the former acts in a DM-independent and -dependent fashion.…”

HLA-DMA Polymorphisms Differentially Affect MHC Class II Peptide Loading

“…The use of FP to study MHCII peptide exchange and release has been widely described (26,28). In this study, FP was used to assess the ability of HLA-DM variants to catalyze peptide exchange under different conditions, and to estimate kinetic parameters of this exchange.…”

“…In addition, a cDNA sequence encoding a flexible Gly-Ser linker and the CLIP 103-117 fragment spaced by a thrombin cleavage site was fused to the N-terminus of the b-chains of the same constructs (DR1C, DR2C, and DR4C). HLA-DM (HLA-DMA1*0101 and HLA-DMB1*0101), described in Nicholson et al (26), was also cloned into the pFastBacDual vector (DMA*0101 and DMB*0101). The original C-terminal protein C tag in the b-chain was replaced with a biotin acceptor sequence, and the a-chain FLAG-tag was kept and used for purification.…”