Interdomain flexibility and interfacial integrity of β-lactamase inhibitory protein (BLIP) modulate its binding to class A β-lactamases |
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Authors: | Liwen Huang Pui-Kin So Yu Wai Chen Yun-Chung Leung Zhong-Ping Yao |
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Institution: | 1.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;2.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 |
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Abstract: | 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. |
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Keywords: | β -lactamases β -lactamase inhibitory protein (BLIP) interdomain flexibility interfacial integrity hydrogen deuterium exchange mass spectrometry molecular dynamics simulation |
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