CHARACTERIZATION OF THE INTERACTIONS BETWEEN MHC CLASS-I SUBUNITS - ASYSTEMATIC-APPROACH FOR THE ENGINEERING OF HIGHER AFFINITY VARIANTS OF BETA(2)-MICROGLOBULIN

Human beta(2)m (h beta(2)m) binds to murine MHC I molecules with highe r affinity than does murine beta(2)m and therefore can be used as a mo del system to define and dissect the interactions between beta(2)m and MHC I heavy chains that promote the stability of the complex. In the present study we compare three-dimensional crystal structures of human and murine MHC I molecules and use functional studies of chimeric hum an:murine beta(2)m variants to define a region of beta(2)m that is inv olved in the higher affinity of h beta(2)m for murine MHC I heavy chai ns. Further examination of the three-dimensional structure in this reg ion revealed conformational differences between human and murine beta( 2)m that affect the ability of an aspartic acid residue at position 53 (D53) conserved in both beta(2)ms to form an ionic bond with arginine residues at positions 35 and 48 of the heavy chain. Mutation of resid ue D53 to either asparagine (D53N) or valine (D53V) largely abrogated the stabilizing effects of h beta(2)m on murine MHC I expression in a predictable manner. Based on this observation a variant of h beta(2)m was engineered to create an ionic bond between the heavy chain and bet a(2)m This variant stabilizes cell surface H-2D(d) heavy chains to a g reater extent than wild-type h beta(2)m. Studying these interactions i n light of the growing database of MHC I crystal structures should all ow the rational design of higher affinity h beta(2)m variants for use in novel peptide-based vaccines capable of inducing cell-mediated immu ne responses to viruses and tumors.