Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology Centre, 138602, Singapore; NTU Institute of Structural Biology, Nanyang Technological University, 636921, Singapore.
School of Biological Sciences, Nanyang Technological University, 637551, Singapore; Yong Loo Lin School of Medicine, National University of Singapore, 117456, Singapore.
J Struct Biol. 2020 Nov 1;212(2):107610. doi: 10.1016/j.jsb.2020.107610. Epub 2020 Sep 2.
Bacterial two-component regulatory systems (TCS) play important roles in sensing environmental stimuli and responding to them by regulating gene expression. VbrK/VbrR, a TCS in Vibrio parahaemolyticus, confers resistance to β-lactam antibiotics through activating a β-lactamase gene. Its periplasmic sensor domain was previously suggested to detect β-lactam antibiotics by direct binding. Here, we report a crystal structure of the periplasmic sensing domain of VbrK (VbrK) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrK is a monomer using size exclusion chromatography coupled with multi-angle light scattering. This observation is also supported by molecular dynamics simulations. To quantify the binding affinity of β-lactam antibiotics to VbrK, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrK did not show any significant binding to β-lactam antibiotics. Therefore, we propose that the detection of β-lactam antibiotics by VbrK is likely to be indirect via an as yet unidentified mechanism.
细菌双组分调控系统（TCS）在感应环境刺激并通过调节基因表达来响应刺激方面发挥着重要作用。副溶血弧菌中的 VbrK/VbrR 是一种 TCS，通过激活β-内酰胺酶基因来赋予对β-内酰胺类抗生素的抗性。其周质感应结构域先前被认为通过直接结合来检测β-内酰胺类抗生素。在这里，我们使用基于硫的单波长异常衍射（S-SAD）相位测定法报告了 VbrK（VbrK）的周质感应结构域的晶体结构。与大多数形成二聚体的细菌感应结构域相反，我们使用尺寸排阻色谱法结合多角度光散射表明 VbrK 是单体。分子动力学模拟也支持这一观察结果。为了定量测定β-内酰胺类抗生素与 VbrK 的结合亲和力，我们进行了等温滴定量热法和其他生物物理分析。出乎意料的是，VbrK 与β-内酰胺类抗生素没有表现出任何显著的结合。因此，我们提出 VbrK 检测β-内酰胺类抗生素可能是通过尚未确定的机制间接进行的。
