Kryger G, Harel M, Giles K, Toker L, Velan B, Lazar A, Kronman C, Barak D, Ariel N, Shafferman A, Silman I, Sussman J L
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Acta Crystallogr D Biol Crystallogr. 2000 Nov;56(Pt 11):1385-94. doi: 10.1107/s0907444900010659.
Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.
本文报道了重组野生型人乙酰胆碱酯酶及其E202Q突变体与束丝菌素-II形成的复合物的结构。束丝菌素-II是一种从东部绿曼巴蛇（Dendroaspis angusticeps）毒液中纯化得到的“三指”多肽毒素。野生型酶复合物的结构通过分子置换法解析，分辨率达到2.8 Å，起始模型为加州电鳐乙酰胆碱酯酶与束丝菌素-II形成的复合物结构，并通过以小鼠乙酰胆碱酯酶的类似复合物为起始模型进行了验证。其整体结构与加州电鳐酶和束丝菌素-II形成的复合物惊人地相似，正如预期的那样，也与小鼠乙酰胆碱酯酶复合物相似。E202Q突变体复合物的结构则以相应的野生型人乙酰胆碱酯酶结构为起始模型进行优化，使用收集到的分辨率为2.7 Å的数据集。两种结构的比较表明，从蛋白质核心去除带电基团并将其替换为中性等排体部分不会破坏活性中心的功能结构。这种结构的一个元素被认为是一个氢键网络，包括Glu202、Glu450、Tyr133残基以及两个桥连水分子，在其他脊椎动物乙酰胆碱酯酶以及人源酶中都是保守的。目前的研究结果与以下观点一致，即该网络的主要作用是使Glu202羧酸盐相对于催化三联体正确定位，从而确定其在乙酰胆碱酯酶与底物和抑制剂相互作用中的功能作用。
