Adamalysin II, a 24 kDa zinc endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of ...mosaic haemorrhagic proteins from various snake venoms. Homologous domains have recently been detected in multimodular mammalian reproductive tract proteins. The 2.0 A crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active‐site cleft separating a relatively irregularly folded subdomain from the calcium‐binding main molecular body composed of a five‐stranded beta‐sheet and four alpha‐helices. The folding of the peptide fragment containing the zinc‐binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin, but is identical to that found in astacin, with the three histidines and a water molecule (linked to the glutamic acid) likewise constituting the zinc ligand; adamalysin II lacks a fifth (tyrosine) zinc ligand, however, leaving its zinc ion tetrahedrally co‐ordinated. Furthermore, adamalysin II and astacin share an identical active‐site basement formed by a common Metturn. Due to their virtually identical active‐site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the astacins (and presumably also the matrix metalloproteinases/mammalian collagenases and the Serratia proteinase‐like large bacterial proteinases) might be grouped into a common superfamily with distinct differences from the thermolysin family.
The cDNAs of several snake venom zinc endopeptidases code for a putative propeptide, which includes the conserved cysteine-containing sequence PKMCGVT. It has been suggested that binding of the ...cysteine thiol function to the active-site zinc, resulting in inactivation of the catalytic domain, occurs in a mode similar to the ‘cysteine switch’ mechanism proposed for matrix metalloproteinases. In order to confirm this hypothesis, inhibition kinetics have been performed on the metalloproteinase adamalysin II of the venom of the snake
Crotalus adamanteus using several cysteine peptides. Among these the synthetic hexapeptide PKMCGV-NH
2, corresponding to the conserved sequence portion of the known propeptides, was found to be by far the strongest inhibitor of this proteinase with a
K
i of 3.4 μM. The inhibitory potencies of an equivalent peptide with the
l-Cys replaced by a
d-Cys or by an
l-Ser as well as of reduced glutathione, cysteine and two unrelated cysteine peptides were by one to two orders of magnitudes lower. These findings strongly support a cysteine switch-like mechanism even for activation of the snake venom metalloproteinases.
Adamalysin II, alias proteinase II, a 24 kDa zinc-endopeptidase isolated from the snake venom of the Eastern diamondback rattlesnake Crotalus adamanteus, is a prototype of the proteolytic domain of ...snake venom metalloproteinases and of domains found in mammalian reproductive tract proteins. Its 2.0 A crystal and molecular structure was solved by multiple isomorphous replacement using six heavy-atom derivatives, and was refined to a crystallographic R-value of 0.172. 201 of the 203 amino acid residues of adamalysin II are defined by electron density; only the first two residues are disordered and crystallographically undefined in the crystal structure. Three-quarters of these crystallographic amino acid residue assignments were confirmed by chemical sequencing. In addition, the active-site zinc-ion, a hepta-coordinated calcium ion, a fixed sulphate anion and 173 solvent molecules were localized in the structure. Adamalysin II is an ellipsoidal molecule with a relatively flat active-site cleft separating the "upper" main body from a small "lower" subdomain. The regularly folded N-terminal upper domain consists essentially of a central, highly twisted five-stranded beta-pleated sheet flanked by a long and a short surface located helix on its convex side, and by two long helices, one of which represents the central "active site helix", on its concave side. The lower subdomain, comprising the last 50 residues, is organized in multiple turns, with the chain ending in a long C-terminal helix and an extended segment clamped to the upper domain via a disulphide bridge. The catalytic zinc-ion, located at the bottom of the active-site cleft, is almost tetrahedrally co-ordinated by His142, His146 and His152, and a water molecule anchored to an intermediate glutamic acid residue (Glu143), with the three imidazole N epsilon 2 nitrogen atoms 2.1 A and the solvent oxygen atom 2.4 A away from the zinc ion. His142, Glu143 and His146 are part of the long active-site helix, which extends up to Gly149, where it turns sharply away towards His152. The importance of these residues for structure and activity of adamalysin II explains their occurrence in the HEXXHXXGXXH consensus sequence. Asp153, which is strictly conserved in these snake venom and reproductive tract metalloproteinases, is buried in the subdomain and seems to stabilize the hydrophobic active-site basement. Some residues behind, the adamalysin peptide chain folds into a characteristic 1,4-turn (the "Met-turn") containing the conserved Met166, which forms a hydrophobic basement for the three zinc-binding imidazoles.
Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian ...reproductive tract proteins, and tumor necrosis factor α convertase, TACE, involved in the release of the inflammatory cytokine, TNFα. The structure of adamalysin II in noncovalent complex with two small‐molecule right‐hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X‐ray diffraction data up to 2.6 and 2.8 Å resolution. The inhibitors bind to the ‐side of the proteinase, inserting between two protein segments, establishing a mixed parallel‐antiparallel three‐stranded β‐sheet and coordinate the central zinc ion in a bidentate manner via their two C‐terminal oxygen atoms. The proteinase‐inhibitor complexes are described in detail and are compared with other known structures. An adamalysin‐based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.
Human plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated ...that antithrombin III, C1-inhibitor, and alpha 2-antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 1-antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino-terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy-terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis--one near the amino-terminal end and another in the exposed reactive site loop of the inhibitor.
A proteinase from Crotalus adamanteus venom has been isolated to the stage of electrophoretic homogeneity by chromatography of the crude venom on CM-Sepharose, Phenyl-Sepharose, Ultrogel AcA 44 and ...DEAE-Sepharose. The proteinase accounts for 100% of the detectable hemorrhagic activity in the venom, and the name C. adamanteus proteinase H is suggested. Proteinase H is a single chain glycoprotein with a molecular weight of 85,700. Proteinase H is active on casein and hide powder azure, but does not digest benzoyl-L-arginine ethyl ester or benzoyl-L-tyrosine ethyl ester. Proteolytic and hemorrhagic activity were both abolished by treatment with EDTA and neither activity was restored by prolonged dialysis against Zn2+ or Ca2+. However, both activities were retained after treatment with phenylmethylsulfonyl fluoride. A minimal hemorrhagic response was elicited in mice by s.c. injection of 0.1 microgram of proteinase H. The caseinolytic activity of proteinase H was not inhibited during incubation with human alpha 2-macroglobulin, nor was the inhibitor inactivated by proteinase H.
Fractionation of opossum (Didelphis virginiana) serum with (NH4)2SO4, followed by chromatography on DEAE-Sepharose, phenyl-Sepharose, and Mono Q HR 5/5, has resulted in the isolation in homogeneous ...condition of a metalloproteinase inhibitor designated oprin (opossum proteinase inhibitor). Oprin is a single-chain glycoprotein (26% carbohydrate) with an estimated Mr = 52,000, pI = 3.5, and E(1%/1 cm) = 11. Oprin inhibited snake venom metalloproteinases, but showed no activity on venom serine proteinases or on bacterial metalloproteinases. Incubation of Crotalus atrox alpha-proteinase (EC 3.4.24.1) with oprin, and analysis of the reaction products by chromatography on Mono Q HR 5/5 and by electrophoresis under nondenaturing conditions, indicated formation of an inactive enzyme/inhibitor complex. The complex dissociated during SDS/polyacrylamide gel electrophoresis. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for part of the open reading frame for oprin were isolated. The cDNA inserts contained nucleotide sequences corresponding to two internal amino acid sequences of oprin which had been separately determined by protein sequence analysis. Protein database screening using a 211 amino acid sequence deduced from one of the cDNA inserts showed no significant homology to known proteinase inhibitors. There was, however, a 36% identity with human alpha 1B-glycoprotein, a plasma protein of unknown function related to the immunoglobulin supergene family. In addition, the amino-terminal sequence of oprin showed 46% identity with human alpha 1B-glycoprotein in a 26 amino acid residue overlap.
The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the ...reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro-binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1'. An adamalysin-based model of tumor necrosis factor-alpha-converting enzyme (TACE) reveals a smaller S1' pocket for this enzyme.
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