FAST ASSOCIATION RATES SUGGEST A CONFORMATIONAL CHANGE IN THE MHC CLASS-I MOLECULE H-2D(B) UPON PEPTIDE BINDING

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum(ER). For this binding reaction, when per formed 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. W e 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 bindi ng of a high-affinity peptide. (ii) With the peptide SV-324-332 (seque nce 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 mo del of binding. (iii) In contrast, binding of this peptide proceeds mu ch faster, with 1.4 x 10(6) M-1 s(-1). These ''mismatch kinetics'' sug gest that peptide binding occurs in several steps, most Likely via a c onformational rearrangement of the peptide binding groove. The structu re of the peptide-class I complex at the time-point of peptide recogni tion may therefore be different from the equilibrium crystal structure s. (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 stu dies.