Structural and functional insight into pan-endopeptidase inhibition by α2-macroglobulins

Abstract

Peptidases must be exquisitely regulated to prevent erroneous cleavage and one control is provided by protein inhibitors. These are usually specific for particular peptidases or families and sterically block the active-site cleft of target enzymes using lock-and-key mechanisms. In contrast, members of the +1400-residue multi-domain α2-macroglobulin inhibitor family (α2Ms) are directed against a broad spectrum of endopeptidases of disparate specificities and catalytic types, and they inhibit their targets without disturbing their active sites. This is achieved by irreversible trap mechanisms resulting from large conformational rearrangement upon cleavage in a promiscuous bait region through the prey endopeptidase. After decades of research, high-resolution structural details of these mechanisms have begun to emerge for tetrameric and monomeric α2Ms, which use 'Venus-flytrap' and 'snap-trap' mechanisms, respectively. In the former, represented by archetypal human α2M, inhibition is exerted through physical entrapment in a large cage, in which preys are still active against small substrates and inhibitors that can enter the cage through several apertures. In the latter, represented by a bacterial α2M from Escherichia coli, covalent linkage and steric hindrance of the prey inhibit activity, but only against very large substrates.

Conformational Differences between Functional Human Immunodeficiency Virus Envelope Glycoprotein Trimers and Stabilized Soluble Trimers

“…2B and C and Table 1). One of the two cross-linked lysine pairs with C ␣ -C ␣ distances beyond 26 Å included Lys 1372, which is immediately adjacent to a conformationally flexible alpha-2-macroglobulin region susceptible to proteolytic cleavage (Table 1) (60,61). These results support the validity of the alpha-2-macroglobulin structure and indicate that our XL-MS methodology can effectively assess the relevance of a structural model.…”

“…tional changes between subunits of alpha-2-macroglobulin are required for its function (60). A single mass spectrometry run identified 22 nonredundant cross-linked peptides in bovine alpha-2-macroglobulin (see Table S1 in the supplemental material).…”

Multiple Architectures and Mechanisms of Latency in Metallopeptidase Zymogens

“…Due to the irreversibility of peptide bond cleavage by MPs in vivo-exceptions have been reported in vitro 25 these enzymes must be exquisitely regulated to prevent aberrant activity, which commonly leads to pathology. Control of MPs is achieved through transcriptional regulation 26 , cellular compartmentalization 27 and specific or broad-spectrum protein inhibitors 28 , among which multi-domain α2-macroglobulins block prey peptidases through pan-peptidase Venusflytrap or snap-trap mechanisms [29][30][31][32] . Other inhibitor examples are the tissue inhibitors of metalloproteinases 33,34 ; the bacterial bifunctional serine-and metalloproteinase inhibitor sermetstatin 35 ; the insect metallopeptidase inhibitor 25 ; and several metallocarboxypeptidase inhibitors 21,36 .…”

A structure-derived snap-trap mechanism of a multispecific serpin from the dysbiotic human oral microbiome

“…The periplasmic location of miropin contrasts with that of most animal serpins, which are secreted and soluble (32). This paradox is reminiscent of ␣ 2 M family inhibitors, whose animal members are also generally secreted to the circulation (38), whereas bacterial members undergo post-translational modifications for periplasmic location similar to miropin (56).…”

“…Generally, serpins target in a highly specific manner certain chymotrypsin-like and/or subtilisin-like SEPs, as well as CEPs, from the papain, cathepsin, and caspase families (31,32,35,36). These inhibitors are one of three covalent suicide inhibitor families (37), which also include the ␣ 2 -macroglobulins (␣ 2 Ms; family I39) (38) and the relatives of baculovirus p35 protein (family I50). Serpins behave like pseudo-substrates, and inhibition is initiated by formation of the Michaelis complex, which occurs without significant structural changes in either enzyme or serpin (39).…”