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^SD) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrK^SD 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^SD, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrK^SD 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.

细菌双组分调节系统 (TCS) 在感知环境刺激并通过调节基因表达对其作出反应方面发挥着重要作用。VbrK/VbrR 是副溶血性弧菌中的一种 TCS，通过激活 β-内酰胺酶基因赋予对 β-内酰胺抗生素的抗性。先前建议其周质传感器结构域通过直接结合检测 β-内酰胺抗生素。在这里，我们使用基于硫的单波长反常衍射 (S-SAD) 定相报告了 VbrK (VbrK ^SD )周质传感域的晶体结构。与大多数形成二聚体的细菌传感器结构域相反，我们表明 VbrK ^SD是一种单体，使用尺寸排阻色谱与多角度光散射相结合。这一观察结果也得到了分子动力学模拟的支持。为了量化 β-内酰胺抗生素对 VbrK ^SD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 ^SD没有显示出与 β-内酰胺抗生素的任何显着结合。因此，我们认为 VbrK 对 β-内酰胺抗生素的检测可能是通过一种尚未确定的机制间接进行的。