An Allosteric Mechanism Controls Antigen Presentation by the H-2KbComplex

Abstract

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) Biochemistry35, 14841−14848], the interactions among the heavy chain, β₂-microglobulin (β₂m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with β₂m 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-chain binding site, in turn, increased markedly the affinity of the heavy chain for β₂m. Binding of peptide variants of the ovalbumin sequence (257−264) to the heavy chain/β₂m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147−155 to the H-2K^d heavy chain/β₂m heterodimer [(3.0 ± 1.0) × 10^-6 M^-1 s^-1 at 37 °C]. The slow phase was a result of both the ternary complex assembly from the “free” heavy chain, β₂m, and peptide as well as an intramolecular conformational transition within the heavy chain/β₂m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or β₂m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of β₂m dissociation from the H-2K^b ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the K^b heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their K^d/nucleoprotein peptide counterparts.