Crystal Structure of the AmpR Effector Binding Domain Provides Insight into the Molecular Regulation of Inducible AmpC β-Lactamase
Crystal Structure of the AmpR Effector Binding Domain Provides Insight into the Molecular Regulation of Inducible AmpC β-Lactamase
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DOI：
10.1016/j.jmb.2010.05.040
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发表时间：
2010-07-30
影响因子：
5.6
通讯作者：
Mark, Brian L.
中科院分区：
文献类型：
--
作者：
Balcewich, Misty D.;Reeve, Thomas M.;Mark, Brian L.
AmpC -内酰胺酶（AmpC）的过量产生是革兰氏阴性菌（如铜绿假单胞菌和肠杆菌科）对青霉素和头孢菌素耐药的一个强大机制。AmpC的表达受lysr型转录调节剂AmpR的调控。ampR和ampC基因形成一个具有重叠启动子的发散操纵子，ampR结合并调节两个基因的转录。AmpR通过与1,6-无氢- n -乙酰muramyl肽家族的一个成员结合诱导ampC，这些肽是在β -内酰胺挑战期间积累的肽聚糖的细胞质分解产物。为了深入了解AmpR调控的结构，我们在1.83埃分辨率下测定了froundii Citrobacter AmpR的效应结合域（effector binding domain， EBD）的晶体结构。AmpR EBD是二聚体，每个单体包括两个采用α / β罗斯曼样折叠的子结构域。位于单体亚结构域之间的是一个口袋，被发现与结晶缓冲分子2-(N-morpholino)乙磺酸结合。该口袋与沿亚结构域I表面的凹槽一起，形成了一个假定的效应结合位点，其中可以模拟1,6-无氢- n -乙酰muramyl五肽分子。在结构域间口袋底部的氨基酸替换被发现使AmpR不能诱导ampC （Thr103Val， Ser221Ala和Tyr264Phe）或导致组成型ampC表达（Gly102Glu）。虽然阻止ampC诱导的取代并没有改变AmpR EBD的整体结构，但圆二色光谱显示，非保守性Gly102Glu突变影响了EBD的二级结构，证实了先前的研究结果，即Gly102Glu诱导构象变化导致本构ampC的产生。(C) 2010 Elsevier Ltd.版权所有。
Hyperproduction of AmpC beta-lactamase (AmpC) is a formidable mechanism of resistance to penicillins and cephalosporins in Gram-negative bacteria such as Pseudomonas aeruginosa and Enterobacteriaceae. AmpC expression is regulated by the LysR-type transcriptional regulator AmpR. ampR and ampC genes form a divergent operon with overlapping promoters to which AmpR binds and regulates the transcription of both genes. AmpR induces ampC by binding to one member of the family of 1,6-anhydro-N-acetylmuramyl peptides, which are cytosolic catabolites of peptidoglycan that accumulate during beta-lactam challenge. To gain structural insights into AmpR regulation, we determined the crystal structure of the effector binding domain (EBD) of AmpR from Citrobacter freundii up to 1.83 angstrom resolution. The AmpR EBD is dimeric and each monomer comprises two subdomains that adopt alpha/beta Rossmann-like folds. Located between the monomer subdomains is a pocket that was found to bind the crystallization buffer molecule 2-(N-morpholino)ethanesulfonic acid. The pocket, together with a groove along the surface of subdomain I, forms a putative effector binding site into which a molecule of 1,6-anhydro-N-acetylmuramyl pentapeptide could be modeled. Amino acid substitutions at the base of the interdomain pocket either were found to render AmpR incapable of inducing ampC (Thr103Val, Ser221Ala and Tyr264Phe) or resulted in constitutive ampC expression (Gly102Glu). While the substitutions that prevented ampC induction did not alter the overall AmpR EBD structure, circular dichroism spectroscopy revealed that the nonconservative Gly102Glu mutation affected EBD secondary structure, confirming previous work suggesting that Gly102Glu induces a conformational change to result in constitutive AmpC production. (C) 2010 Elsevier Ltd. All rights reserved.