Staphylococcus aureus Sortase A transpeptidase -: Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop
Staphylococcus aureus Sortase A transpeptidase -: Calcium promotes sorting signal binding by altering the mobility and structure of an active site loop
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DOI：
10.1074/jbc.m506123200
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发表时间：
2006-01-20
影响因子：
4.8
通讯作者：
Clubb, RT
中科院分区：
文献类型：
--
作者：
Naik, MT;Suree, N;Clubb, RT
革兰氏阳性菌中的许多毒力因子通过分选酶（一组广泛分布的半胱氨酸转肽酶）共价锚定至细胞壁肽聚糖。金黄色葡萄球菌分选酶A蛋白（SrtA）是分选酶家族的原型成员，并被Ca 2+激活，这是一种适应，可促进宿主定植，因为在人体组织中遇到浓度升高的该离子。在这里，我们表明，一个单一的Ca 2+离子绑定到SrtA上的有序口袋变构激活催化，通过调节一个大的活性位点环的结构和动力学。详细的氮-15弛豫测量表明，Ca 2+可以促进自适应识别的基板通过诱导缓慢的微毫秒的时间尺度动态的活性位点。有趣的是，弛豫补偿Carr-Purcell-Meiboom- Gill实验表明，这些运动的时间尺度与离子结合直接相关。定点诱变的结果表明，这种运动偶联是由Glu-171的侧链介导的，Glu-171位于β 6/β 7环内，并显示有助于Ca 2+结合。可用的结构和动力学数据与Ca 2+激活的环闭合模型兼容，其中β 6/β 7环在由Ca 2+稳定的结合活性封闭形式和从活性位点去除关键底物接触残基的开放的高度柔性状态之间波动。
Many virulence factors in Gram-positive bacteria are covalently anchored to the cell-wall peptidoglycan by sortase enzymes, a group of widely distributed cysteine transpeptidases. The Staphylococcus aureus Sortase A protein (SrtA) is the archetypal member of the Sortase family and is activated by Ca2+, an adaptation that may facilitate host colonization as elevated concentrations of this ion are encountered in human tissue. Here we show that a single Ca2+ ion bound to an ordered pocket on SrtA allosterically activates catalysis by modulating both the structure and dynamics of a large active site loop. Detailed nitrogen-15 relaxation measurements indicate that Ca2+ may facilitate the adaptive recognition of the substrate by inducing slow micro- to millisecond time-scale dynamics in the active site. Interestingly, relaxation compensated Carr-Purcell-Meiboom- Gill experiments suggest that the time scale of these motions is directly correlated with ion binding. The results of site-directed mutagenesis indicate that this motional coupling is mediated by the side chain of Glu-171, which is positioned within the beta 6/beta 7 loop and shown to contribute to Ca2+ binding. The available structural and dynamics data are compatible with a loop closure model of Ca2+ activation, in which the beta 6/beta 7 loop fluctuates between a binding competent closed form that is stabilized by Ca2+, and an open, highly flexible state that removes key substrate contacting residues from the active site.