Assembly and dissociation of human leukocyte antigen (HLA)-A2 studied by real-time fluorescence resonance energy transfer

Class I major histocompatibility complex (MHC) heterodimer, composed of hum an leukocyte antigen (HLA)-A2 heavy chain and human beta(2)-microglobulin ( beta(2)m), was produced by denaturation and gel filtration of the recombina nt water-soluble HLA-A2/beta(2)m/peptide ternary complex in 8 M urea Tris-H Cl buffer, followed by refolding of the separated chains without peptide. P eptide affinity and kinetics of the ternary complex formation and dissociat ion were investigated in real time by monitoring the fluorescence resonance energy transfer (FRET) from intrinsic HLA-A2 heavy-chain tryptophans to a dansyl fluorophore conjugated to the bound peptide. Peptide binding to the heterodimer was a second order process with rate constants linearly depende nt upon temperature in Arrhenius coordinates over 0-20 degrees C. The bindi ng rate constant of pRT6C-dansyl [ILKEPC(dansyl)HGV] at 37 degrees C evalua ted by extrapolation of the Arrhenius plot was (2.0 +/- 0.5) x 10(6) M-1 s( -1). Association of the heavy chain with beta(2)m was a first order process , apparently controlled by a conformational transition in the heavy chain. One of these conformations bound to beta(2)m to form the heavy chain/beta(2 )m heterodimer whereas the second conformer oligomerized. Peptide dissociat ion from the ternary complex was a first-order reaction over the temperatur e range 20-37 degrees C, suggesting that the ternary complex also exists in two conformations. Taken together, the present data suggest that associati on of beta(2)m changes the HLA-A2 heavy-chain conformation thereby promotin g peptide binding. Peptide dissociation from the ternary complex induces di ssociation of the heavy-chain/beta(2)m heterodimer thereby causing oligomer ization of the heavy chain. The lability of the HLA-A2/beta(2)m heterodimer and the strong tendency of the "free" heavy chain to oligomerize may provi de an efficient mechanism for control of antigen presentation under physiol ogical conditions by reducing the direct loading of HLA with exogenous pept ide at the cell surface.