Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations.
Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations.
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DOI：
10.1016/j.jmgm.2016.04.006
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发表时间：
2016-06
影响因子：
2.9
通讯作者：
Wereszczynski J
中科院分区：
文献类型：
--
作者：
Shrestha P;Wereszczynski J
在革兰氏阳性菌中，索糖酶是关键的毒力因素。这些酶通过转肽反应将表面蛋白嵌入细胞壁，该反应涉及识别目标蛋白中的五肽“分类信号”，将其切割，并将其共价连接到第二种底物上，该底物随后插入细胞壁。尽管研究得很好，但排序酶执行这些功能的机制的几个方面仍然不清楚。特别是，实验揭示了两个潜在的分选信号结合基序：“苏氨酸输出”(Thr-Out)结构和“苏氨酸-输入”(Thr-in)结构，其中催化关键苏氨酸残基突出到溶液中，苏氨酸残基插入结合位点。为了确定哪一种状态是生物相关的，我们对结合在分选信号底物上的金黄色葡萄球菌索拉酶A(SrtA)酶进行了一系列常规和混合量子力学/分子力学(QM/MM)分子动力学模拟。通过使用多维的元动力学方法，我们的模拟既能够绘制出SrtA在Thr-In和Thr-Out状态下的酰化机理，也能够确定这些反应的自由能最小值和势垒。结果表明，两种状态的催化机理相似，但Thr-In构型的自由能垒较低，表明Thr-In是催化相关状态。这对于促进我们对索酸酶作用机制的了解，以及我们未来基于结构的药物设计工作都具有重要的意义，目的是抑制体内的索酸酶功能。
Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide “sorting signal” in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a “Threonine-Out” (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a “Threonine-In” (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus Sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo.