Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Department of Crystallography and Structural Biology, Institute of Physical Chemistry "Rocasolano", CSIC , 28006 Madrid, Spain.
J Am Chem Soc. 2017 Feb 8;139(5):2102-2110. doi: 10.1021/jacs.6b12565. Epub 2017 Jan 27.
The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the β-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three β-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a-each with the β-lactam in the allosteric site-defined (with preceding PBP2a structures) as the "open" or "partially open" PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known "closed" apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for β-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.
β-内酰胺类抗菌药物的作用机制是使催化其肽聚糖细胞壁交联步骤的酶发生功能上不可逆的酰化。革兰氏阳性病原体金黄色葡萄球菌使用一种主要的耐药机制。一种称为青霉素结合蛋白 2a（PBP2a）的酶被引入到这个生物合成途径中以完成交联。PBP2a 有效地将β-内酰胺类抗生素作为潜在抑制剂区分开来，并有利于肽聚糖底物。这种区分的基础是一个变构位点，远离活性位点，当被适当占据时，它会同时打开活性位点内的守门员残基，并重新排列关键残基的构象以允许催化。我们使用晶体学研究和计算分析来解决这种调节的分子基础。三种β-内酰胺（苯唑西林、头孢吡肟、头孢他啶）与 PBP2a 的复合物的晶体结构-每种β-内酰胺都位于变构位点，定义为“开放”或“部分开放”的 PBP2a 状态。在靠近活性位点的一个特定环运动被确定为伴随活性位点打开的活性位点构象变化的驱动力。为了确定信号通路，将这种环运动与变构位点的效应物结合相关联，是通过计算来完成的，参考了已知的“封闭”无配体 PBP2a X 射线晶体结构状态。这种相关性使我们能够对与 PBP2a 初始肽聚糖底物结合、酰化酶形成以及酰基转移到第二个肽聚糖底物以达到交联相吻合的结构进行计算模拟。这些研究为变构位点与活性位点之间的通信以及β-内酰胺对肽聚糖底物的模拟提供了重要的见解，这是对新兴 PBP2a 耐药突变的机制理解的基础。
