Role of Sug1, a 19S proteasome ATPase, in the transcription of MHC I and the atypical MHC II molecules, HLA-DM and HLA-DO.

The 19S proteasome regulatory particle plays a critical role in cellular proteolysis. However, emerging evidence suggests roles for 19S proteasome subunits in regulating yeast and mammalian transcription. It has been previously shown that Sug1 is important for the transcription of MHC II molecules. We report here that Sug1 also has a role in regulating transcription of class I MHC and the MHC II-like molecules, HLA-DM and HLA-DO. Reduction of Sug1 expression causes a decrease in the transcription of MHC I and MHC II-like molecules. In addition, we show that association of Sug1 with MHC promoters is followed by the recruitment of the CREB-binding protein (CBP) and the class II transactivator (CIITA). Reduction of Sug1 expression is accompanied by decreased recruitment of CBP and CIITA to the MHC promoters and decreased histone H3 acetylation in these promoters. These studies suggest that Sug1 plays a critical role in transcription of MHC class I, and the MHC class II-like molecules, HLA-DM and HLA-DO.

Conformational lability in the class II MHC 310 helix and adjacent extended strand dictate HLA-DM susceptibility and peptide exchange.

HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3(10) helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.

Model for the peptide-free conformation of class II MHC proteins.

BACKGROUND: Major histocompatibility complex proteins are believed to undergo significant conformational changes concomitant with peptide binding, but structural characterization of these changes has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here we use molecular dynamics simulations and experimental probes of protein conformation to investigate the peptide-free state of class II MHC proteins. Upon computational removal of the bound peptide from HLA-DR1-peptide complex, the alpha50-59 region folded into the P1-P4 region of the peptide binding site, adopting the same conformation as a bound peptide. Strikingly, the structure of the hydrophobic P1 pocket is maintained by engagement of the side chain of Phe alpha54. In addition, conserved hydrogen bonds observed in crystal structures between the peptide backbone and numerous MHC side chains are maintained between the alpha51-55 region and the rest of the molecule. The model for the peptide-free conformation was evaluated using conformationally-sensitive antibody and superantigen probes predicted to show no change, moderate change, or dramatic changes in their interaction with peptide-free DR1 and peptide-loaded DR1. The binding observed for these probes is in agreement with the movements predicted by the model. CONCLUSION/SIGNIFICANCE: This work presents a molecular model for peptide-free class II MHC proteins that can help to interpret the conformational changes known to occur within the protein during peptide binding and release, and can provide insight into possible mechanisms for DM action.

A polymorphic pocket at the P10 position contributes to peptide binding specificity in class II MHC proteins.

Peptides bind to class II major histocompatibility complex (MHC) proteins in an extended conformation. Pockets in the peptide binding site spaced to accommodate peptide side chains at the P1, P4, P6, and P9 positions have been previously characterized and help to explain the obtained peptide binding specificity. However, two peptides differing only at P10 have significantly different binding affinities for HLA-DR1. The structure of HLA-DR1 in complex with the tighter binding peptide shows that the peptide binds in the usual polyproline type II conformation, but with the P10 residue accommodated in a shallow pocket at the end of the binding groove. HLA-DR1 variants with polymorphic residues at these positions were produced and found to exhibit different side chain specificity at the P10 position. These results define a new specificity position in HLA-DR proteins.

A hairpin turn in a class II MHC-bound peptide orients residues outside the binding groove for T cell recognition.

T cells generally recognize peptide antigens bound to MHC proteins through contacts with residues found within or immediately flanking the seven- to nine-residue sequence accommodated in the MHC peptide-binding groove. However, some T cells require peptide residues outside this region for activation, the structural basis for which is unknown. Here, we have investigated a HIV Gag-specific T cell clone that requires an unusually long peptide antigen for activation. The crystal structure of a minimally antigenic 16-mer bound to HLA-DR1 shows that the peptide C-terminal region bends sharply into a hairpin turn as it exits the binding site, orienting peptide residues outside the MHC-binding region in position to interact with a T cell receptor. Peptide truncation and substitution studies show that both the hairpin turn and the extreme C-terminal residues are required for T cell activation. These results demonstrate a previously unrecognized mode of MHC-peptide-T cell receptor interaction.

Exploration of the P6/P7 region of the peptide-binding site of the human class II major histocompatability complex protein HLA-DR1.

Crystal structures of the class II major histocompatibilty complex (MHC) protein, HLA-DR1, generally show a tight fit between MHC and bound peptide except in the P6/P7 region of the peptide-binding site. In this region, there is a shallow water-filled pocket underneath the peptide and between the pockets that accommodate the P6 and P7 side chains. We investigated the properties of this pocket with the idea of engineering substitutions into the corresponding region of peptide antigens to increase their binding affinity for HLA-DR1. We investigated d-amino acids and N-alkyl modifications at both the P6 and P7 positions of the peptide and found that binding of peptides to HLA-DR1 could be increased by incorporating an N-methyl substitution at position 7 of the peptide. The crystal structure of HLA-DR1 bound to a peptide containing a P7 N-methyl alanine was determined. The N-methyl group orients in the P6/P7 pocket, displacing one of the waters usually bound in this pocket. The structure shows that the substitution does not alter the conformation of the bound peptide, which adopts the usual polyproline type II helix. An antigenic peptide carrying the N-methyl modification is taken up by antigen-presenting cells and loaded onto endogenous class II MHC molecules for presentation, and the resultant MHC-peptide complexes activate antigen-specific T-cells. These results suggest a possible strategy for increasing the affinity of weakly immunogenic peptides that might be applicable to the development of vaccines and diagnostic reagents.