Institut Pasteur, Unité d'Immunologie Structurale, CNRS, URA 2185, Département de Biologie Structurale et Chimie, F-75015 Paris, France.
J Struct Biol. 2010 Mar;169(3):360-9. doi: 10.1016/j.jsb.2009.10.010. Epub 2009 Oct 24.
Ammodytoxin A (AtxA) and its natural isoform AtxC from the venom of Vipera ammodytes ammodytes belong to group IIA-secreted phospholipases A(2) which catalyze the hydrolysis of glycerophospholipids and exhibit strong neurotoxic and anticoagulant effects. The two isoforms, which differ in sequence by only two amino acid residues (Phe124>Ile and Lys128>Glu), display significant differences in toxicity and anticoagulant properties and act on protein targets including neurotoxic proteic receptors and coagulation factor Xa with significantly different strengths of binding. In order to characterize the structural basis of these functional differences, we have determined the crystal structures of the two isoforms. Comparison of the structures shows that the mutation Lys128>Glu in AtxC could perturb interactions with FXa, resulting in lower anticoagulant activity, since the side chain of Glu128 is partly buried, making a stabilizing hydrogen bond with the main-chain nitrogen atom of residue Thr35. This interaction leads to a displacement of the main polypeptide chain at positions 127 and 128 (identified by mutagenesis as important for interaction with FXa), and a different orientation of the side chain of unmutated Lys127. The mutation Phe124>Ile in AtxC induces no significant conformational changes, suggesting that the differences in toxicity of the two isoforms are due essentially to differences in surface complementarity in the interaction of the toxin with the neurotoxic protein receptor. The crystal structures also reveal a novel dimeric quaternary association involving significant hydrophobic interactions between the N-terminal alpha-helices of two molecules of ammodytoxin related by crystallographic symmetry. Interactions at the dimer interface include important contributions from Met7, which is unique to ammodytoxin. Equilibrium sedimentation experiments are consistent with the crystallographic model. Competition experiments using SPR technology show complete inhibition of AtxA binding to FXa by calmodulin (CaM). The crystal structure shows that the C-terminal region, important for binding to FXa and CaM, is fully exposed and accessible for interaction with proteic receptors in both the monomeric and dimeric forms of ammodytoxin described here.
氨昆毒素 A (AtxA) 及其天然同工型 AtxC 来自 Vipera ammodytes ammodytes 的毒液，属于 IIA 组分泌型磷脂酶 A(2)，可催化甘油磷脂水解，并具有强烈的神经毒性和抗凝作用。这两种同工型仅在两个氨基酸残基（Phe124>Ile 和 Lys128>Glu）上存在序列差异，在毒性和抗凝特性上表现出显著差异，并作用于包括神经毒性蛋白受体和凝血因子 Xa 的蛋白靶标，其结合强度有显著差异。为了表征这些功能差异的结构基础，我们确定了这两种同工型的晶体结构。结构比较表明，AtxC 中的突变 Lys128>Glu 可能会破坏与 FXa 的相互作用，导致抗凝活性降低，因为 Glu128 的侧链部分被掩埋，并与残基 Thr35 的主链氮原子形成稳定的氢键。这种相互作用导致主多肽链在位置 127 和 128 处发生位移（通过突变确定为与 FXa 相互作用的重要位置），并且未突变的 Lys127 的侧链具有不同的取向。AtxC 中的突变 Phe124>Ile 不会引起明显的构象变化，这表明两种同工型的毒性差异主要归因于毒素与神经毒性蛋白受体相互作用时表面互补性的差异。晶体结构还揭示了一种新的二聚体四级缔合，涉及通过晶体学对称性相关的两个氨昆毒素分子的 N 端 α-螺旋之间的显著疏水相互作用。二聚体界面上的相互作用包括独特的 Met7 的重要贡献。平衡沉降实验与晶体学模型一致。使用 SPR 技术的竞争实验表明，钙调蛋白 (CaM) 完全抑制 AtxA 与 FXa 的结合。晶体结构表明，对于与 FXa 和 CaM 的结合至关重要的 C 端区域在单体和二聚体形式的氨昆毒素中完全暴露并可与蛋白受体相互作用。
