Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole
Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole
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DOI：
10.1074/jbc.m113.509273
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发表时间：
2014-03-28
影响因子：
4.8
通讯作者：
Clubb, Robert T.
中科院分区：
文献类型：
--
作者：
Jacobitz, Alex W.;Wereszczynski, Jeff;Clubb, Robert T.
背景：分选酶催化展示细菌表面蛋白的转肽反应。结果：结构和计算研究揭示了分选酶B酶如何识别其分选信号底物。结论：分选酶使用底物稳定的含氧阴离子孔催化转肽。重要性：分选酶半胱氨酸转肽酶通过共价键将蛋白质连接到细菌细胞壁或组装纤维状皮利，促进细菌粘附。该酶超家族的成员广泛分布于革兰氏阳性菌中，这些革兰氏阳性菌经常利用多种分选酶来加工其肽聚糖。分选酶使用保守的活性位点His-Cys-Arg三联体催化转肽，所述三联体将位于其蛋白质底物的C末端的分选信号连接到位于细胞表面上的氨基亲核试剂。然而，尽管广泛的研究，底物识别的催化机制和分子基础仍然知之甚少。在这里，我们报告的晶体结构的金黄色葡萄球菌分选酶B酶的共价复合物与其NPQTN分选信号底物的类似物，揭示了结构基础，通过它显示的IsdC蛋白参与血红素铁清除人类血红蛋白。计算建模、分子动力学模拟和靶向氨基酸诱变的结果表明，Glu（224）的主链酰胺和Arg（233）的侧链在分选酶B中形成氧阴离子空穴，其稳定高能四面体催化中间体。令人惊讶的是，结合的分选信号底物内的高度保守的苏氨酸残基通过经由氢键合稳定活性位点精氨酸残基的位置来促进含氧阴离子孔的构建。分子动力学模拟和一级序列保守性表明，分选信号稳定的含氧阴离子孔是分选酶超家族中酶的普遍特征。
Background: Sortase enzymes catalyze a transpeptidation reaction that displays bacterial surface proteins. Results: Structural and computational studies reveal how the sortase B enzyme recognizes its sorting signal substrate. Conclusion: Sortase enzymes catalyze transpeptidation using a substrate-stabilized oxyanion hole. Significance: The results of this work could facilitate the rational design of sortase inhibitors.Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu(224) and the side chain of Arg(233) form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.