The Structure of the C949S Mutant Human α2-Macroglobulin Demonstrates the Critical Role of the Internal Thiol Esters in Its Proteinase-Entrapping Structural Transformation

Abstract

A three-dimensional reconstruction of a protein-engineered mutant α2-macroglobulin (α2M) in which a serine residue was substituted for the cysteine 949 (C949S), making it unable to form internal thiol ester moieties, was compared with native and methylamine-transformed α2Ms. The native α2M structure consists of two oppositely oriented Z-shaped strands. Thiol ester cleavage following an encounter with a proteinase or a nucleophilic attack by methylamine causes a structural transformation in which the strands assume an opposite handedness and a significant portion of the protein density migrates from the distal ends of the molecule toward the center. The C949S mutant showed a protein density distribution very similar to that of transformed α2M, with a compact central region of protein density connected to two receptor-binding arms on each end of the molecule. Since no particle shapes characteristic of native or half-transformed α2Ms were seen in electron micrographs and the C949S mutant and α2M–methylamine structures are highly similar, we conclude that the intact thiol esters maintain native α2M in a quasi-stable state. In their absence, α2M folds into the more stable transformed structure, which displays the functionally important receptor-binding domains and contains the proteinase-entrapping internal cavity.