Elucidating mechanisms of beta-lactam antibiotic resistance through serial crystallography and molecular simulations

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Bacterial antibiotic resistance is a global public health emergency, already responsible for >1.2 m deaths per annumworldwide with up to 10 m predicted by 2050. Beta-lactams (penicillins and related drugs) are the most widely usedantibiotics. In Gram-negative bacteria such as Escherichia coli (the leading cause of bloodstream infections in the U.K.)beta-lactam resistance is usually due to beta-lactamase enzymes that cleave the amide bond in the beta-lactam ring andabolish antibiotic activity.This proposal applies state-of-the-art approaches in X-ray crystallography and computational simulations of chemicalreactions to study the mechanism(s) by which beta-lactamases degrade beta-lactams, information that will guidedevelopment of small molecule inhibitors that block their activity and restore beta-lactam effectiveness against beta-lactamase producing bacteria. X-ray crystallography provides near-atomic resolution structural information on proteins,and their interactions with small molecules, but is traditionally a static technique unable to describe transientlypresent species or capture dynamic information on e.g. interconversion of states in chemical reactions. Recentlydeveloped serial methods, where reactions in micron-scale crystals are initiated by rapid mixing or light-dependent chemistry and a single image per crystal subsequently collected after a defined time interval, can however nowyield structural information on the millisecond time scale. This offers potential to create "molecular movies" composed ofa series of structural snapshots at different time points along a reaction pathway, for the first time enabling structuraldescriptions of transient, mechanistically important states in enzyme-catalysed reactions. In this project we will applyserial techniques, working together with scientists at Diamond Light Source, to investigate reactions of beta-lactamases with their antibiotic substrates and with inhibitor candidates. Structures that we obtain will be used inmolecular simulations to understand the dynamic properties of enzyme bound species and to investigate theenergetics of their interconversion. Simulations based on classical (Newtonian) mechanics will investigate theconformational flexibility of individual states along a reaction pathway, while quantum mechanical methods will enableus to calculate the energy barriers controlling their interconversion and so assess the reactivity of individual complexes.In combination, these two approaches will reveal how different beta-lactamases employ different mechanisms to degrade common substrates, and how and why different enzymes vary in their reactivity varies towards different antibioticsand susceptibility towards different inhibitors. This information will be valuable in identifying how beta-lactams maybe modified to evade beta-lactamase activity, and how beta-lactamase inhibitors may be optimised for activity againstthe widest range of beta-lactamase targets.