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Structural Characterization of Myotoxic Ecarpholin S From Echis carinatus Venom

Xingding Zhou ^*, Tien-Chye Tan ^*, S. Valiyaveettil , Mei Lin Go , R. Manjunatha Kini ^*, Adrian Velazquez-Campoy  and J. Sivaraman ^*

^* Department of Biological Sciences, Department of Chemistry, and Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117543, Singapore; and Institute of Biocomputation and Physics of Complex Systems, Fundacion Aragon I + D, University of Zaragoza, Zaragoza 50009, Spain

Correspondence: Address reprint requests to J. Sivaraman, Dept. of Chemistry, Faculty of Science, National University of Singapore, Singapore 117543, Singapore. E-mail: dbsjayar{at}nus.edu.sg.

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. Ecarpholin S, isolated from the venom of the snake Echis carinatus sochureki, is a phospholipase A₂ (PLA₂) belonging to the Ser⁴⁹-PLA₂ subgroup. It has been characterized as having low enzymatic but potent myotoxic activities. The crystal structures of native ecarpholin S and its complexes with lauric acid, and its inhibitor suramin, were elucidated. This is the first report of the structure of a member of the Ser⁴⁹-PLA₂ subgroup. We also examined interactions of ecarpholin S with phosphatidylglycerol and lauric acid, using surface plasmon resonance, and of suramin with isothermal titration calorimetry. Most Ca²⁺-dependent PLA₂ enzymes have Asp in position 49, which plays a crucial role in Ca²⁺ binding. The three-dimensional structure of ecarpholin S reveals a unique conformation of the Ca²⁺-binding loop that is not favorable for Ca²⁺ coordination. Furthermore, the endogenously bound fatty acid (lauric acid) in the hydrophobic channel may also interrupt the catalytic cycle. These two observations may account for the low enzymatic activity of ecarpholin S, despite full retention of the catalytic machinery. These observations may also be applicable to other non-Asp⁴⁹-PLA₂ enzymes. The interaction of suramin in its complex with ecarpholin S is quite different from that reported for the Lys⁴⁹-PLA₂/suramin complex_, where the interfacial recognition face (i-face), C-terminal region, and N-terminal region of ecarpholin S play important roles. This study provides significant structural and functional insights into the myotoxic activity of ecarpholin S and, in general, of non-Asp⁴⁹-PLA₂ enzymes.