Abstract
The crystal structure of TM-1, a small snake-venom metalloproteinase (SVMP) isolated from Taiwan habu (Trimeresurus mucrosquamatus), was determined at 1.84 Å resolution with resultant R and Rfree values of 0.179 and 0.216, respectively. The overall structure of TM-1 is an oblate ellipsoid that contains three disulfide crosslinks, Cys119-Cys198, Cys160-Cys182 and Cys162-Cys165. The overall structure of TM-1 contains one zinc ion which is bound to four ligands, including three conserved histidines and one water molecule, displaying a tetrahedral geometry. We find that the distinct active site structures between TM-1 and TM-3 may reflect the different sensitivities toward the endogenous inhibitors from structural comparison of TM-1 and TM-3. Moreover, we have modeled the three endogenous inhibitors, i.e., pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) and pyroGlu-Lys-Trp (pEKW), into the active site of TM-1 structure. Results from interaction analysis of TM-1 and inhibitors show that the P-2 site of inhibitors is important for binding to TM-1 via hydrogen bonds and hydrophobic interactions. By proteolysis experiments of TM-1 using oxidized insulin B-chain as substrate, we sugguest P-1 and P-2 sites of substrates are important for cleavage by TM-1. The preferential amino acids at P-1 site are Leu, Phe and His, while The preferential amino acids at P-2 site are Leu, Val and Tyr. In conclusions, our results indicate that the structural difference between TM-1 and TM-3 may influence their endogenous inhibitor sensitivities and substrate specificity. Additionally, we also suggest that the P-1 and P-2 sites of substrate are critical for cleavage by TM-1.