Bailey Melanie L, Shilton Brian H, Brandl Christopher J, Litchfield David W
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Biochemistry. 2008 Nov 4;47(44):11481-9. doi: 10.1021/bi800964q. Epub 2008 Oct 10.
The catalytic domain of the peptidyl-prolyl cis/ trans isomerase Pin1 is a member of the FKBP superfold family. Within its active site are two highly conserved histidine residues, H59 and H157. Despite their sequence conservation in parvulin PPIase domains, the role of these histidine residues remains unclear. Our previous work (Behrsin et al. (2007) J. Mol. Biol. 365, 1143- 1162.) was consistent with a model where one or both histidines had critical roles in a hydrogen bonding network in the active site. Here, we test this model by looking at the effect of mutations to H59 and H157 on Pin1 function, activity, and protein stability. Using a yeast complementation assay, we show that both H59 and H157 can be mutated to non-hydrogen bonding residues and still support viability. Surprisingly, a nonfunctional H59L mutation can be rescued by a mutation of H157, to leucine. This double mutation (H59L/H157L) also had about 5-fold greater isomerase activity than the H59L mutation with a phosphorylated substrate. Structural analyses suggest that rescue of function and activity results from partial rescue of protein stability. Our findings indicate that H59 and H157 are not required for hydrogen bonding within the active site, and in contrast to the active site C113, they do not participate directly in catalysis. Instead, we suggest these histidines play a key role in domain structure or stability.
肽基脯氨酰顺反异构酶Pin1的催化结构域是FKBP超折叠家族的成员。其活性位点内有两个高度保守的组氨酸残基，即H59和H157。尽管它们在小脯氨酸异构酶结构域中序列保守，但这些组氨酸残基的作用仍不清楚。我们之前的研究工作（Behrsin等人，《分子生物学杂志》，2007年，第365卷，第1143 - 1162页）与一种模型相符，即一个或两个组氨酸在活性位点的氢键网络中起关键作用。在此，我们通过研究H59和H157突变对Pin1功能、活性和蛋白质稳定性的影响来测试该模型。使用酵母互补试验，我们发现H59和H157都可以突变为非氢键形成残基，并且仍能支持细胞存活。令人惊讶的是，无功能的H59L突变可以通过将H157突变为亮氨酸来挽救。这种双突变（H59L/H157L）对于磷酸化底物的异构酶活性也比H59L突变高约5倍。结构分析表明，功能和活性的挽救源于蛋白质稳定性的部分挽救。我们的研究结果表明，活性位点内的氢键形成并不需要H59和H157，并且与活性位点的C113不同，它们不直接参与催化作用。相反，我们认为这些组氨酸在结构域结构或稳定性中起关键作用。
