Huang Liwen, So Pui-Kin, Chen Yu Wai, Leung Yun-Chung, Yao Zhong-Ping
State Key Laboratory of Chemical Biology and Drug Discovery, Research Institute for Future Food and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region, China; State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation) and Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Institute of The Hong Kong Polytechnic University, Shenzhen, China.
State Key Laboratory of Chemical Biology and Drug Discovery, Research Institute for Future Food and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region, China.
J Biol Chem. 2021 Aug;297(2):100980. doi: 10.1016/j.jbc.2021.100980. Epub 2021 Jul 21.
β-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αβ domains conjugated by an interdomain loop and can effectively bind and inactivate class A β-lactamases, which are responsible for resistance of bacteria to β-lactam antibiotics. The varied ability of BLIP to bind different β-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A β-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A β-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.
β-内酰胺酶抑制蛋白（BLIP）由通过结构域间环连接的αβ结构域串联重复组成，能够有效结合并灭活A类β-内酰胺酶，而这类酶会导致细菌对β-内酰胺抗生素产生耐药性。目前对于BLIP结合不同β-内酰胺酶的能力差异以及单点突变显著增强BLIP变体的结构决定因素了解甚少。在此，我们研究了BLIP与三种临床常见的A类β-内酰胺酶（TEM1、SHV1和PC1）结合时的构象动力学，其解离常数在亚纳摩尔至微摩尔之间。氢氘交换质谱显示，与TEM1强结合时，结构域间区域的灵活性显著受到抑制，但与SHV1或PC1弱结合时则无显著变化。结构域间区域的E73M和K74G突变分别提高了对SHV1和PC1的结合亲和力，与野生型相比，结构域间区域的灵活性显著增加，且结合时构象变化有利。相反，在这些BLIP突变体的游离态和结合态中均观察到界面环135 - 145的刚性增加。同样，BLIP的分子动力学模拟显示，在所有复合物中，环135 - 145的灵活性都发生了剧烈变化。我们的结果首次表明，结构域间连接子更高的灵活性以及界面环135 - 145更高的刚性，可能是增强BLIP对A类β-内酰胺酶抑制作用的理想决定因素。总之，这些发现为增强抑制剂的设计提供了独特的见解。
