Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

Abstract

We have characterized four human ␣ 2 -macroglobulin (␣ 2 M) 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 ␣ 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 "cavity body" seen in the low resolution x-ray structure of transformed ␣ 2 M. Inhibition of proteinases by humanresults from a series of conformational changes that are initiated by the proteinase and that result in the physical sequestration of the proteinase within the closed cage-like structure of the conformationally altered ␣ 2 M (1, 2). The initiating event is a proteolytic cleavage by the attacking proteinase near the center of the ␣ 2 M polypeptide in a region termed the bait region (3, 4). Cleavage anywhere within the bait region also results in activation of an internal thiol ester toward cleavage by nucleophiles. From studies on the kinetics of cleavage of the thiol ester by nucleophiles and of the subsequent conformational change within the ␣ 2 M, it has been shown that the conformational change occurs cooperatively after both thiol esters within one half of the ␣ 2 M tetramer have been cleaved (5, 6).Knowledge of the structural relationship between the thiol ester and the bait region and hence of the details of the activation mechanism is limited by the absence of a high resolution x-ray structure of either native or conformationally altered human ␣ 2 M. From fluorescence resonance energy transfer measurements, it was shown that the four cysteines that form the four thiol esters of the human ␣ 2 M tetramer are centrally located and are about 35 Å apart (7). This was subsequently confirmed by a low resolution (ϳ10 Å) x-ray structure of conformationally altered ␣ 2 M (8). NMR measurements on ␣ 2 M showed that the bait region of each monomer was unusually flexible (9, 10) and that it lies close (10 -17 Å) to the cysteine of the thiol ester in the transformed protein (11). However, the location of the four bait regions is still not known. Studies from this laboratory on ␣ 2 M varian...

α-Macroglobulins Are Present in Some Gram-negative Bacteria

“…The second-order rate constant calculated for the reaction of methylamine with the thiol ester was 11.5 M Ϫ1 s Ϫ1 . This compares with a value of 12.5 M Ϫ1 s Ϫ1 determined here for the thiol ester in human ␣ 2 M reacted with methylamine, which is closely similar to values determined elsewhere under similar conditions (35)(36)(37). Thus ECAM appears to contain an intact thiol ester that is protected from nucleophilic attack, even though the YY motif present in other ␣-macroglobulins does not seem to be present.…”

On the Structural Changes of Native Human α2-Macroglobulin upon Proteinase Entrapment

“…Since new inter and intramolecular disulfide bonds were formed in a variant that contained a single cysteine residue within the bait region, it was proposed that the four bait domains are located in close proximity in the center of the molecule (36). However, the location of the new disulfide bonds in the amino acid sequence was not determined so that it is equally possible that they involve residues outside the bait regions and consequently their location and putative proximity remains ambiguous.…”

Hypochlorite-induced structural modifications enhance the chaperone activity of human α2-macroglobulin

“…It has previously been reported that oxidation of 14 methionine residues and a single tryptophan residue results in the dissociation of tetrameric α 2 M into dimers (25). The exact positions of these residues have yet to be identified; however, we predict the central part of each α 2 M subunit containing the bait region (i.e., the target for proteolytic cleavage located between amino acid residues 666 and 706 in human α 2 M) to be important given that point mutations within the bait region have been shown to disrupt the noncovalent binding of α 2 M dimers (63). The bait region of human α 2 M is rich in methionine residues (i.e., M666, M673, M688, M697, and M713 located on each human α 2 M monomer; the latter is located just beyond the bait region), and in the intact tetramer, these methionine residues are closely aligned at the interface between the noncovalently associated α 2 M dimers (64).…”