Thiol ester role in correct folding and conformation of human α2‐macroglobulin

Abstract

To determine the role of the thiol ester in the folding of human α₂-macroglobulin (α₂M) in the active conformation, we have characterized a recombinant variant of α₂M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S α₂M behaves like methylamine-treated plasma α₂M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric α₂M does not require a pre-formed thiol ester. Active α₂M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S α₂M followed by thiol ester formation and a conformational change that gives the native active state.