Pritchard L, Dufton M J
Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, Scotland.
J Mol Biol. 1999 Jan 29;285(4):1589-607. doi: 10.1006/jmbi.1998.2437.
The structural and functional evolution of the Kunitz/bovine pancreatic trypsin inhibitor (BPTI) family of proteins, which includes serine proteinase inhibitors and potassium channel blockers, was analysed with the evolutionary trace method. This method highlights sites in aligned primary sequences whose side-chain variation can be strongly linked with the past development of different functional classes or subgroups within the family. A total of 16 such "class-specific" positions distributed throughout the molecular fold were identified. On average, the side-chain chemistry at these positions had been more conserved and made greater contribution to molecular stability than the side-chain chemistry at remaining sites of variation. It was possible to use these 16 positions to describe the division of the Kunitz/BPTI family into general functional classes. According to known complexes of inhibitor variants with serine proteinases, only two of the 16 class-specific positions appear to be directly involved in intermolecular recognition via the "antiproteinase site". Instead, from various critical locations in the fold, the remainder seem to have been associated with various degrees of intramolecular conformational adjustment to the underlying framework of the antiproteinase site. It is, therefore, implied that functional diversification in this family has been founded upon both sustained evolutionary selection and conformational adjustment. The findings are important for protein engineers wishing to alter the binding selectivity of these molecules, because it appears that the issue of target recognition is dependent on the conformation of the chain segment to which the interactive side-chains are attached. To retarget members of this family towards potentially novel peptide binding sites, substitutions at certain structurally significant class-specific positions could be a good starting point.
采用进化追踪法分析了Kunitz/牛胰蛋白酶抑制剂（BPTI）蛋白家族的结构和功能演变，该家族包括丝氨酸蛋白酶抑制剂和钾通道阻滞剂。这种方法突出了比对后的一级序列中的位点，其侧链变异与家族内不同功能类别或亚组的过去发展密切相关。总共确定了分布在整个分子折叠结构中的16个这样的“类别特异性”位置。平均而言，这些位置的侧链化学性质比其余变异位点的侧链化学性质更保守，对分子稳定性的贡献更大。利用这16个位置可以描述Kunitz/BPTI家族划分为一般功能类别的情况。根据抑制剂变体与丝氨酸蛋白酶的已知复合物，16个类别特异性位置中只有两个似乎通过“抗蛋白酶位点”直接参与分子间识别。相反，从折叠结构的各个关键位置来看，其余位置似乎与抗蛋白酶位点的基础框架进行了不同程度的分子内构象调整有关。因此，这意味着该家族的功能多样化是基于持续的进化选择和构象调整。这些发现对希望改变这些分子结合选择性的蛋白质工程师很重要，因为似乎目标识别问题取决于与相互作用侧链相连的链段的构象。为了使该家族成员靶向潜在的新型肽结合位点，在某些具有结构重要性的类别特异性位置进行替换可能是一个很好的起点。
