Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform? Helena C. Castro a;b; *, Dione M. Silva a , Charles Craik b , Russolina B. Zingali a a Departamento de Bioqu| ¨mica Me ¨dica-ICB, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil b Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA Received 27 October 2000; received in revised form 30 January 2001; accepted 23 February 2001 Abstract The Lachesis muta thrombin-like enzyme (LM-TL) is a single chain serine protease that shares 38% sequence identity with the serine protease domain of thrombin and also displays similar fibrinogen-clotting activity. In addition, the 228 amino acid residue LM-TL is 52% identical to trypsin, and cleaves chromogenic substrates with similar specificity. Herein we report a three-dimensional (3D) model validated experimentally for LM-TL based on these two homologous proteins of known 3D structure. Spatial modeling of LM-TL reveals a serine protease with a chymotrypsin fold presenting a hydrophobic pocket on its surface, involved in substrate recognition, and an important 90's loop, involved in restricting the LM-TL catalytic site cleft. Docking analysis showed that LM-TL would not form a stable complex with basic pancreatic trypsin inhibitor and wild-type ecotin since its 90's loop would restrict the access to the catalytic site. LM-TL formed acceptable interactions with fibrinopeptide A and a variant of ecotin ; ecotin-TSRR/R in which both the primary and secondary binding sites are mutated Val81Thr, Thr83Ser, Met84Arg, Met85Arg and Asp70Arg. Furthermore, analysis of the primary structures of LM-TL and of the seven snake venom thrombin-like enzymes (SVTLEs) family reveals a subgroup formed by LM-TL, crotalase, and bilineobin, both closely related to thrombin. Therefore, LM-TL provides an initial point to compare SVTLEs with their counterparts, e.g. the mammalian serine proteases, and a basis for the localization of important residues within the little known SVTLEs family. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Serine protease; Three-dimensional model; Snake venom; Thrombin; Trypsin; Ecotin 1. Introduction The Lachesis muta thrombin-like enzyme (LM-TL) is a serine protease puri¢ed from L. muta venom, which preferentially cleaves Arg^Gly bonds in ¢brin- ogen K chains [1]. This clotting enzyme (228 residues) releases ¢brinopeptide A in the conversion of ¢brin- ogen to ¢brin, analogous to thrombin, but hydro- lyzes synthetic substrates with speci¢city similar to trypsin [2]. As expected, the amino acid sequence of this serine protease is 38 and 52% identical to thrombin and trypsin [3], respectively. The putative 0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S0167-4838(01)00177-7 Abbreviations : LM-TL, Lachesis muta thrombin-like enzyme ; SVTLE, snake venom thrombin-like enzyme ; TSV-PA, plasmino- gen activator from Trimeresurus stejenegeri venom; BPTI, bovine pancreatic trypsin inhibitor ; SCR, structurally conserved region ; PDB, Protein Data Bank; RMS, root-mean-square ; ecotin- TSRR/R, ecotin with mutations in both primary (Val81Thr, Thr83Ser, Met84Arg, and Met85Arg) and secondary binding sites (Asp70Arg) ; ecotin-TSRR, ecotin with mutations in primary binding site (Val81Thr, Thr83Ser, Met84Arg, and Met85Arg) ; FRE, ¢brinogen recognition exosite ; u-PA, urokinase-type plas- minogen activator * Corresponding author. Fax: +55-21-2708647; E-mail : hcastro@bioqmed.ufrj.br Biochimica et Biophysica Acta 1547 (2001) 183^195 www.bba-direct.com