Prizant Maya, Eisenberg-Domovich Yael, Hytönen Vesa P, Kulomaa Markku S, Wilchek Meir, Bayer Edward A, Livnah Oded
Department of Biological Chemistry, The Institute of Life Sciences, The Wolfson Centre for Applied Structural Biology; The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
J Mol Biol. 2006 May 5;358(3):754-63. doi: 10.1016/j.jmb.2006.02.044. Epub 2006 Mar 3.
The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudo-substrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction.
研究了一系列抗生物素蛋白衍生物对生物素对硝基苯酯（BNP）的水解作用。令人惊讶的是，一种超耐热抗生物素蛋白相关蛋白（AVR4）表现出非凡却令人费解的水解活性。为了评估促成该反应的分子决定因素，将AVR4的晶体结构与抗生物素蛋白、链霉抗生物素蛋白以及这两种蛋白与生物素对硝基苯胺（BNA，BNP的惰性酰胺类似物）形成复合物的关键突变体的晶体结构进行了比较。结构显示，一个关键的赖氨酸残基对抗生物素蛋白介导的BNP水解有作用，但对AVR4介导的反应贡献较小。实际上，不同抗生物素蛋白之间的各自水解速率反映了几个分子参数，包括结合位点结构、配体与溶剂的可及性以及配体的构象和随之而来的对有效亲核攻击的敏感性。在抗生物素蛋白中，BNP与Lys111的相互作用以及L3,4环的无序状态（以及随之而来的溶剂可及性）共同构成了水解背后的主要驱动力，而在AVR4中，配体（假底物）的状态是一个主要区别特征。在后者蛋白中，L3,4环的独特构象限制了假底物，从而使羰基碳原子暴露于亲核攻击。此外，由于其构象，AVR4复合物中的假底物无法与保守的赖氨酸类似物（Lys109）相互作用；相反，该功能被与Arg112的极性相互作用所取代。结果表明，在高度相似的蛋白质中，不同的残基可以执行相同的功能，并且此类蛋白质活性位点结构的细微差异可导致不同的反应模式。
