| In order to protect the host from pathogenic microorganisms, cells of the adaptive immune response must be alerted to the presence of an infectious agent. In vertebrates, the cellular immune response is critically dependent on the generation of "epitopes" or pathogen-derived peptide fragments bound to major histocompatibility complex II (MHCII) molecules. Recognition of peptide/MHCII complexes through T cell receptor (TCR) engagement leads to CD4+ T lymphocyte activation, acquisition of effector function, and clearance of the pathogen.; To investigate this model, we examined the contribution of minor anchor residues to the selection of peptide via the MHCII pathway. Using the binding of a peptide derived from influenza hemagglutinin to the human MHCII allele HLA-DR1 (DR1) as a model system, we show that affinity and DM stability can be influenced by peptide side chain substitutions at more highly solvent exposed positions in the peptide/MHCII interface. Multiple substitutions reduce affinity to a level greater than the loss of a buried anchor interaction. Biochemical data and structural modeling suggest that these effects may be due to disruption of the hydrogen bond network between MHCII and bound peptide.; The analysis of multiply substituted peptides also revealed cooperativity in peptide/MHCII interactions. We hypothesized that this cooperative effect was related to conformational changes that accompany peptide/MHCII binding. Analysis of a complete set of peptides encompassing all possible combinations of substitutions demonstrates cooperativity in affinity and complex stability. To determine the molecular basis of the cooperative effect, we mutated the DR1 molecule to remove a hydrogen bond and find a reduced level of cooperativity in affinity and complex stability. These results suggest that the ability of MHCII molecules to bind diverse peptide sequences may be due in part to the presence of conformational changes dependent on hydrogen bond formation.; One of the unresolved issues in the process of epitope selection is the molecular mechanism by which DM is able to catalyze the exchange of peptide from MHCII. Therefore, we asked whether a cooperative model of peptide/MHCII interactions may reveal novel aspects of DM mechanism. Interestingly, in the presence of DM, we find extremely low levels of cooperativity as a function of dissociation rate. (Abstract shortened by UMI.)... |
