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 2M) bait region variants (G679C, M690C, V700C, and T705C) to test the hypothesis that the bait regions are involved in the interface between noncovalently associated dimers. All four variants folded correctly as judged by many normal properties. However, the presence of a cysteine resulted in disulfide formation between otherwise noncovalently associated dimers in all four variants. The extent of disulfide cross-linking varied with the location of the cysteine and gave a mixture of species that 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 crosslinks correlating with blockage of conformational change. The stoichiometry of trypsin inhibition was less in all cases than for wild-type alpha 2M. 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 "cavity body" seen in the low resolution x-ray structure of transformed alpha 2M.
Bowen
M E
ME
Department of Biochemistry and Molecular Biology, University of Illinois at Chicago 60612-4316, USA.
Gettins
P G
PG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D000511:alpha-Macroglobulins; D003545:Cysteine