Localization of the proteinases in the human α2-macroglobulin-chymotrypsin complex by image processing of electron micrographs

Abstract Human alpha 2-macroglobulin (α2M), a large tetrameric plasma glycoprotein, inhibits a wide spectrum of proteinases by a particular “trapping” mechanism resulting from the proteolysis of peptide bonds at specific “bait” regions. This induces the hydrolysis of four thiol esters triggering both the possible covalent bonding of the proteinases and a considerable structural change in the α2M molecule, also observed following direct cleavage of the thiol esters by methylamine. By subtracting average images of electron micrographs from two populations of α2M molecules in the same biochemical state (with both the four cleaved bait regions and thiol esters), but containing either two or zero chymotrypsins, we are able to demonstrate the position of the two proteinases inside the tetrameric α2M molecule. The comparison of the α2M molecules transformed either by immobilized chymotrypsin or methylamine shows that the proteolysis of the bait regions seems of minimal importance for the general shape of the molecule and provides a direct visualization of the actual role of the thiol esters in the conformational change.