Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum(ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2D(b) class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (T-m) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s(-1) and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s(-1) for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s(-1). These "mismatch kinetics" suggest that peptide binding occurs in several steps, most Likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the T-m of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.