Structure of acutolysin-C, a haemorrhagic toxin from the venom of Agkistrodon acutus, providing further evidence for the mechanism of the pH-dependent proteolytic reaction of zinc metalloproteinases

Abstract

The structure of acutolysin-C, a haemorrhagic zinc metalloproteinase from the venom of Agkistrodon acutus, has been analyzed and refined at 2.2 A resolution. The space group of the crystal is P2(1)2(1)2(1), with unit-cell dimensions a = 46.84, b = 49.52, c = 95.34 A. One molecule was found in each asymmetric unit. The phasing problem was solved by the molecular-replacement program AMoRe. Crystallographic refinement was performed using X-PLOR, leading to final R and free R factors of 0.176 and 0.272, respectively. The residue sequence of acutolysin-C was determined mainly by electron density. No density was found for the first residue at the N--terminus and the last two residues at the C-terminus, which was also the case for most other P-I class snake-venom metalloproteinases (SVMPs). Acutolysin-C has two highly conserved characteristic sequences His142-Glu143-X-X-His146-X-X-Gly149-X-X-His15 2 and Cys162-Ile163-Met164. The enzyme has three disulfide bridges: Cys117-Cys195, Cys157-Cys179 and Cys159-Cys162. The entire structure shows good agreement with that of other reported P-I class SVMPs and has two subdomains with a cleft in which one catalytic zinc ion is localized. However, the local conformation (especially the disulfide configurations), the coordination of the catalytic water molecules and some residue side chains differ compared with other P-I class SVMPs. The proteolytic activities of SVMPs are sensitive to the pH value. The molecular superpositions around the proteolytic active sites of all the P-I class SVMP crystal structures show that the distances between the zinc ion and its ligands are not correlated with the crystallization pH values, although the contact distances between the catalytic water molecule and the O atoms of the Glu143 carboxylate group in the neutral and weakly alkaline structures are shorter than those in weakly acidic structures, and the closer the crystallization pH value of one enzyme is to its optimal activity pH value, the shorter the contact distances. Overall, all P-I class SVMPs have similar conformations in the active-site cleft. The size of the active site is not correlated with the crystallization pH values or the proteolytic activities. The disulfide bridge Cys117-Cys195 is conserved in all crystal structures of P-I class SVMPs, whereas the conformation and number of disulfide bridges in the C-terminal subdomain differ. Acutolysin-C has no structural calcium ion, which may not affect the proteolytic activity or haemorrhagic activity directly.

Molecular models of the Mojave rattlesnake (Crotalus scutulatus scutulatus) venom metalloproteinases reveal a structural basis for differences in hemorrhagic activities

“…GP1, GP2, and GP3 contained a second disulfide bond that linked the cysteine residues at positions 102 and 183, and for GP2, at amino acid positions 87 and 187. This second disulfide bond bridged the carboxyl terminus to the amino terminal end of the molecule in the same manner as shown by the crystal structures of rattlesnake metalloproteinases [34,35,[43][44][45]. Unexpectedly, the GP2 model had an additional third disulfide bond (149-171) that pulled the α-helix D towards the methionine-turn loop and which caused α-helix D to be partitioned into two smaller segments (Fig.…”

“…3, contained two major structural domains, an upper N-terminal domain and a lower C-terminal domain. All the molecular models have four conserved a-helices, termed A, B, C, and D, and five N-terminal β-strands (I-V) that form the central hydrophobic β-pleated sheet core region, which is arranged in the same topological conformation as published for the crystal structures of adamalysin II of C. adamanteus [34,35,[43][44][45] and for atrolysin C (Ht-d) of C. atrox [34,35]. The C-terminal domain included a downward loop preceding the Zn +2 binding site but located after α-helix C. All four models had the same conformation for the methionine-turn (Met turn) with the methionine residue located approximately 2Å away from the zinc ion within the zinc binding pocket.…”

“…At the C-terminal domain, the four groups contained two short β-strands that harbor the Met turn. This structural region was arranged in the same order as reported for all RSVMPs [34,35,44,45].…”

“…Over the past decade, several high-resolution crystal structures of RSVMPs have been solved, and these data have enabled structural biologists to gain a deeper understanding of the substrate specificity and selectivity of these toxins [35,[44][45][46][47]. Given the limited number of available snake metalloproteinase crystal structures, predicting the molecular structures of additional RSVMPs by applying molecular modeling techniques is essential for designing catalytic inhibitors that block hemorrhage by these toxins.…”

Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the x-ray structure of adamalysin II and atrolysin C

“…Recently the X-ray structure analysis of the interaction of atrolysin C (form D) with an inhibitor (27) and the structure of acutolysin A and C were published (28)(29)(30). We have also calculated the differential average homology profile for fibrolase compared to other fibrinolytic metzincins and fibrolase compared to other hemorrhagic metzincins.…”