Insights into the molecular interaction of snake venom metalloproteinase, atrolysin C, and tissue inhibitors of metalloproteinases
Pinto A.F.M.; Terra R.M.S.; Nagase H.; Serrano S.M.T; Guimarães J.A. and Fox J.W.
Department of Microbiology, University of Virginia, VA, USA; Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, RS, Brazil
Introduction: Snake venom metalloproteinases (SVMPs) are reprolysis with an active role in the pathogenesis of envenomation. Atrolysin C is a P-I SVMP from Crotalus atrox venom, which efficiently degrades basement membrane proteins producing hemorrhage. Control of proteolytic activity of metalloproteinases upon cellular matrix proteins is exerted, among others, by endogenous proteins, the tissue inhibitors of metalloproteinases (TIMPs). This family of proteins comprises four members which have different specificities to their target enzymes. In this work we evaluated the inhibitory profile of TIMP1, TIMP2 and the N-terminal domain of TIMP3 (NTIMP3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular docking and molecular dynamics simulation.
Methodology: Inhibition of atrolysin C by TIMP1, TIMP2 and NTIMP3 was assayed using azocollagen as substrate. TIMP1, TIMP2 and Atrolysic C coordinates were taken from PDB. A theoretical model of NTIMP3 was built using TIMP1 and TIMP2 as templates. Docking structures of atrolysin C to the TIMPs were predicted using the fully automated ZDOCK server. Molecular dynamics simulations of the complexes were performed using the GROMACS package and the Gromos96 forcefield. The structures were submitted to an energy minimization and then to 2ns molecular simulations. Results were analyses using LigPlot tool according to known biological characteristics.
Results and Conclusions: While TIMP1 and TIMP2 had no inhibitory activity upon atrolysin C, NTIMP3 was a potent inhibitor with a Ki value of approximately 150 nM. Among the obtained complexes, atrolysin C/NTIMP3 was the only one that remained in the predicted inhibitory conformation thus confirming the inhibition studies. This complex was similar to those already described in other MMP-TIMPs crystallographic structures. This study shows for the first time the inhibition of a SVMP by a TIMP and shed light on the structural determinants required for the interaction between these proteins.
Supported by CNPq and CAPES
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