Marchot P, Prowse C N, Kanter J, Camp S, Ackermann E J, Radić Z, Bougis P E, Taylor P
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0636, USA.
J Biol Chem. 1997 Feb 7;272(6):3502-10. doi: 10.1074/jbc.272.6.3502.
Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, DeltaPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.
束丝菌素是一种乙酰胆碱酯酶的选择性肽类抑制剂，是从蛇毒中分离出的三指肽毒素超家族的成员。束丝菌素2（Fas2）-乙酰胆碱酯酶复合物晶体结构的可得性为详细研究这种毒素与其靶位点的相互作用提供了契机。为此，我们构建了一个带有合适前导肽的合成束丝菌素基因，用于在哺乳动物细胞中表达和分泌。在中国仓鼠卵巢细胞中表达并扩增的重组野生型Fas2被纯化至同质，发现其组成和生物学活性与毒液衍生的毒素相同。在复合物晶体结构显示为显著界面接触点或构象决定因素的位置产生了16个突变。环I的两个突变体T8A/T9A和R11Q、最长环II的10个突变体R24T、K25L、R27W、R28D、H29D、缺失Pro30、P31R、K32G、M33A以及V34A/L35A，和环III的两个突变体D45K和K51S，在人胚肾细胞中瞬时表达。基于用多克隆抗Fas2血清对突变体进行滴定，确定了Fas2突变体对小鼠乙酰胆碱酯酶的抑制效力。Arg27、Pro30和Pro31突变体的Fas2活性各自丧失了两个或更多数量级，这表明环II末端的这三个残基子集主导了环构象以及Fas2与酶的相互作用。Arg24、Lys32和Met33突变体丧失了约一个数量级，表明这些残基对复合物强度有适度贡献，而Lys25、Arg28、Val34-Leu35、Asp45和Lys51突变体表现得与Fas2一样具有活性。Thr8-Thr9、Arg11和His29突变体显示出比Fas2更高的抑制活性与免疫化学效价之比。这可能反映了这些区域中的免疫显性决定因素或构象中的分子内重排，从而增强了相互作用。在与乙酰胆碱酯酶界面处的众多Fas2残基中，只有少数几个似乎为复合物的高亲和力提供了实质性的能量贡献。
