Stable peptide binding to MHC class II molecule is rapid and is determined by a receptive conformation shaped by prior association with low affinity peptides

Formation of stable class II MHC/peptide complex involves conformational changes and proceeds via an intermediate. Although this intermediate complex forms and dissociates in minutes, its conversion to a stable complex is a very slow process, taking up to a few days to reach completion. Here, we investigate the different steps of this binding and demonstrate that the conformational changes necessary to generate a receptive molecule is the rate-determining slow step in the process, while formation of the stable MHC/peptide complex is very rapid. With HLA-DR1 as our model class II molecule, we first used low affinity variants of hemagglutinin peptide (HA_306-318), which lack the principal anchor, to shape the conformation of the MHC and then studied the kinetics of stable binding of HA_306-318 to such an induced conformation. We found that the apparent association rate of HA_306-318 is equivalent to the dissociation rate of the low affinity peptide. A 4- to 18-fold enhancement in the binding rates of HA_306-318 was observed depending on the dissociation rates of the low affinity peptides. These results establish that 1) formation of stable MHC/peptide complexes is very rapid and 2) prior binding of low affinity peptide induces a receptive conformation in MHC for efficient stable peptide binding. Furthermore, in the absence of any free peptide, this receptive molecule rapidly reverts to slow binding behavior toward the subsequently offered peptide. These results have important implications for the roles of low affinity MHC/peptide complexes in Ag presentation.