BAIT REGION INVOLVEMENT IN THE DIMER-DIMER INTERFACE OF HUMAN ALPHA(2)-MACROGLOBULIN AND IN MEDIATING GROSS CONFORMATIONAL CHANGE - EVIDENCE FROM CYSTEINE VARIANTS THAT FORM INTERDIMER DISULFIDES

We have characterized four human alpha(2)-macroglobulin (alpha(2)M) ba it region variants (G679C, M690C, V700C, and T705C) to test the hypoth esis that the bait regions are involved in the interface between nonco valently associated dimers, All four variants folded correctly as judg ed by many normal properties, However, the presence of a cysteine resu lted in disulfide formation between otherwise noncovalently associated dimers in all four variants, The extent of disulfide cross-linking va ried with the location of the cysteine and gave a mixture of species t hat probably contained two, one, or zero interdimer disulfides in the tetramer. This was reflected in heterogeneity of conformational change upon thiol ester cleavage by methylamine, with the presence of crossl inks correlating with blockage of conformational change, The stoichiom etry of trypsin inhibition was less in all cases than for wild-type al pha(2)M The M690C variant also showed evidence of some species with an intramolecular disulfide between bait regions of monomers within the same dimer, Taken together, the results are consistent with a location of the four bait regions in contact with, or in very close proximity to, one another, This suggests that they form all or part of the ''cav ity body'' seen in the low resolution x-ray structure of transformed a lpha(2)M.