A Molecular Mechanism for Lys49-Phospholipase A2 Activity Based on Ligand-induced Conformational Change

Andre L.B Ambrosio,M Cristina Nonato,Heloísa S Selistre De Araújo,Raghuvir Arni,Richard J Ward,Charlotte L Ownby,Dulce H.F De Souza,Richard C Garratt

doi:10.1074/jbc.m410588200

Abstract

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)-phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca(2+)-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Highlights

Phospholipases A2 (PLA2,1 EC 3.1.1.4) constitute a large family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of phospholipids, producing free fatty acids and lysophos
An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2؉-independent membrane damaging activity and myotoxicity
In the case of form I, the refinement converged to final residuals of Rfactor ϭ 20.5% and Rfree ϭ 24.7%, based on a model that consists of 121 residues for the polypeptide chain, 78 sites treated as water oxygens, a Ligand-induced Conformational Change in Lys49-PLA2

Summary

Introduction

Phospholipases A2 (PLA2,1 EC 3.1.1.4) constitute a large family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of phospholipids, producing free fatty acids and lysophos-. Lys49-PLA2s were believed to be catalytically inactive because of flipping of the Cys29–Gly peptide bond and their stereochemical incapacity to bind the cofactor calcium ion and stabilize the tetrahedral intermediate observed in the calcium-dependent catalytic reaction promoted by Asp49-PLA2s [31]. This hypothesis was supported by structural analyses that have shown that the ⑀-amino group of Lys is located in the position occupied by Ca2ϩ in Asp49-PLA2 [32,33,34]. The use of refolded recombinant enzyme, where this risk has been eliminated [37], appears to confirm the original hypothesis that Lys49-PLA2s are catalytically inactive

Results

Discussion

Conclusion