Crystal Structure of Myotoxin II, a Monomeric Lys49- Phospholipase A 2 Homologue Isolated from the Venom of Cerrophidion (Bothrops) godmani R. K. Arni,* ,1 M. R. M. Fontes,† C. Barberato,‡ J. M. Gutie ´rrez,§ C. Dı ´az,§ and R. J. Ward ¶ *Department of Physics, IBILCE/UNESP, Sa ˜o Jose ´ do Rio Preto-SP. CP 136, CEP15054000, Brazil; ¶ Department of Chemistry, Faculdade de Filosofia, Cie ˆncias e Letras (FFCLRP), USP, Ribeira ˜ o Preto-SP, Brazil; †Department of Physics and Biophysics, UNESP, Botucatu, Brazil; ‡Faculdades Associadas de Sa ˜ o Paulo, FASP, Sa ˜ o Paulo, Brazil; and §Faculdad de Microbiologı ´a, Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose ´, Costa Rica Received December 12, 1998, and in revised form March 10, 1999 Lys49-Phospholipase A 2 (Lys49-PLA 2 ) homologues damage membranes by a Ca 2 -independent mecha- nism which does not involve catalytic activity. With the aim of determining the structural basis for this novel activity, we have solved the crystal structure of myotoxin-II, a Lys49-PLA 2 isolated from the venom of Cerrophidion (Bothrops) godmani (godMT-II) at 2.8 Å resolution by molecular replacement. The final model has been refined to a final crystallografic residual (R factor ) of 18.8% (R free  28.2%), with excellent stereo- chemistry. godMT-II is also monomeric in the crystal- line state, and small-angle X-ray scattering results demonstrate that the protein is monomeric in solution under fisicochemical conditions similar to those used in the crystallographic studies. © 1999 Academic Press Key Words: snake venoms; Lys49-Phospholipase A 2 ; myotoxins; Cerrophidion (Bothrops) godmani. Phospholipases A 2 (PLA 2 , 2 EC 3.1.1.4) are calcium- dependent enzymes that hydrolyze the sn-2 ester bond of phospholipids, releasing lysophospholipds and fatty acids (1). Ten classes of PLA 2 s have been described (2), of which classes I, II, III, V, and X (secreted 14-kDa PLA 2 s) are abundant in a variety of biological fluids, particularly in pancreatic secretions, inflammatory ex- udates, and in reptile and arthropod venoms (3). PLA 2 s are major components of snake venoms, and in addi- tion to their catalytic activities further demonstrate a broad spectrum of pharmacological functions, such as neurotoxicity, myotoxicity, cardiotoxicity, anticoagu- lant effects, and influences on both platelet aggrega- tion and the inflammatory response (3, 4). The catalytic activity of secreted 14-kDa PLA 2 s is dependent on the binding of Ca 2+ , which serves as a cofactor. Coordination of the Ca 2+ ion in the PLA 2 calcium-binding loop includes an aspartate residue at position 49 which plays a crucial role in the stabiliza- tion of the tetrahedral transition state intermediate in catalytically active PLA 2 s (5). A group of myotoxic PLA 2 s present in the venoms of some species of Agkis- trodon, Bothrops, and Trimeresurus (family Viperidae) are characterized by a Asp to Lys substitution of resi- due 49 (6 –9). This Asp49 to Lys substitution elimi- nates the calcium-binding ability of these Lys49-PLA 2 homologues and, as a consequence, their catalytic ac- tivity is abolished. Despite the lack of enzymatic activ- ity, these Lys49-PLA 2 s induce myotoxic effects (10) and retain the ability to disrupt both synthetic and biolog- ical membranes by a Ca 2+ -independent mechanism of action (11–13). It has been suggested that a molecular region located at the C-terminus, rich in hydrophobic and basic residues, is responsible for membrane pene- tration and destabilization (14, 15, 19). Several Lys49 myotoxic PLA 2 s have been crystal- lized, and the structures of Agkistrodon piscivorus pi- scivorus Lys49 (16), Bothrops asper myotoxin II (BaspTX-II) (17), Bothrops moojeni myotoxin II (MjTX- II) (18), bothropstoxin I from Bothrops jararacussu 1 To whom correspondence should be addressed at Departamento de Fı ´sica, IBILCE/UNESP, Caixa Postal 136. CEP 15054-600, Sa ˜o Jose ´ do Rio Preto-SP, Brazil. Fax: (++55)17 2248692. E-mail: arni@df.ibilce.unesp.br. 2 Abbreviations used: PLA 2 , phospholipase A 2 ; godMT-II, C. god- mani; myotoxin II; SAXS, small-angle X-ray scattering; BthTX-I, B. jararacussu bothropstoxin I; MjTX-II, B. moojeni myotoxin II; BaspTX-II, B. asper myotoxin II; PrTX-I, B. pirajai myotoxin I; rms, root mean square. 0003-9861/99 $30.00 177 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 366, No. 2, June 15, pp. 177–182, 1999 Article ID abbi.1999.1210, available online at http://www.idealibrary.com on