An allosteric mechanism controls antigen presentation by the H-2K(b) complex

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2K(b) molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2K(d) ternary complex [Gakamsky et al, (1996) Biochemistry, 35, 14841-14848], the interactions among the heavy chain, beta(2)-microglobulin (beta(2)m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta(2)m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides, Interaction of peptides with the heavy-c hain binding site, in turn, increased markedly the affinity of the heavy ch ain for beta(2)m. Binding of peptide variants of the ovalbumin sequence (25 7-264) to the heavy chain/beta(2)m heterodimer was found to be a biphasic r eaction. The fast phase was a second-order process with nearly the same rat e constants as those of binding of peptides derived from the influenza viru s nucleoprotein 147-155 to the H-2K(d) heavy chain/beta(2)m heterodimer [(3 .0 +/- 1.0) x 10(-6) M-1 s(-1) at 37 degrees C]. The slow phase was a resul t of both the ternary complex assembly from the "free" heavy chain, beta(2) m, and peptide as well as an intramolecular conformational transition withi n the heavy chain/beta(2)m heterodimer to a peptide binding conformation. B iexponential kinetics of peptide or beta(2)m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta(2)m dissociation from the H-2K(b) ternary compl ex were, in the limits of experimental accuracy, independent of the structu re of the bound peptide, though their affinities differed by an order of ma gnitude. Dissociation of peptides from the K-b heavy chain was always faste r than from the ternary complexes, yet the heavy chain/peptide complexes we re considerably more stable compared with their K-d/nucleoprotein peptide c ounterparts.