Time-resolved structural analysis of the extended spectrum beta-lactamase CTX-M-14

这项建议的目的是用时间分辨连续同步加速器结晶学(TR-SSX)解析超广谱β-内酰胺酶CTX-M-14的反应机理的催化细节。β-内酰胺酶对世界上最常用的抗生素-β-内酰胺类抗生素产生耐药性，从而在细菌感染的治疗中发挥重要作用。CTX-M-14 DT12β-内酰胺酶是从临床相关肺炎克雷伯菌中分离出来的一种细菌，具有广泛的底物谱(ESBL)，不仅能降解青霉素类药物，还能降解头孢菌素和单菌素，从而导致对大量抗生素的耐药性。第一个目标是通过系列同步加速器结晶学(SSX)确定稳定的反应中间体，如Michaelis-Menten络合物、共价中间体和产物络合物的室温结构。这些静态的室温结构为低温结构的比较和时间分辨X射线衍射实验提供了重要的依据。通过tr-SSX，我的目标是解决抗生素催化过程中的动态构象变化，到目前为止，这些构象变化只能来自静态冷冻结构。在反应条件为最慢周转动力学的情况下，我将启动tr-SSX实验。使用先前建立的TR-SSX协议，例如通过将基质溶液的液滴射入芯片安装的微晶体上以引发反应，将能够以短时间间隔记录反应的快照。或者，可以使用光学笼状底物来引发反应。此外，我还将利用最近建立的突发系列数据收集模式，该模式能够以1.35毫秒的时间分辨率快速连续地收集多个时间点。这些短的时间框架将使在催化过程中跟踪底物的结构中间体成为可能。最后，这些实验将得到MD-模拟的补充，旨在连接构象变化的子步骤。

The aim of this proposal is to resolve the catalytic details of the reaction mechanism of the extended spectrum beta-lactamase CTX-M-14 with time-resolved serial synchrotron crystallography (TR-SSX).Beta-lactamase confer resistance to beta-lactam antibiotics, the most commonly prescribed antibiotics in the world, and thereby play an important role in the treatment of bacterial infections. The CTX-M-14 DT12 beta-lactamase is an isolate from the clinically relevant bacterium Klebsiella pneumoniae, which has an extended substrate spectrum (ESBL) and is able to hydrolyze not only penicillins but also cephalosporins and monobactam antibiotics and thus contributes to resistance against a large group of antibiotics. The first aim is to determine the room-temperature structures of stable reaction intermediates like the Michaelis-Menten complex, the covalent intermediate as well as the product complex, via serial synchrotron crystallography (SSX). These statis room-temperature structures display an important basis for the comparison to cryo-structures and time-resolved x-ray diffraction experiments. Via TR-SSX I aim to resolve resolve the dynamic conformational changes during catalysis of the antibiotics, which so far can only be derived from static cryo-structures. With the reaction conditions for the slowest turnover kinetics I will initiate TR-SSX experiments. Using the previously established TR-SSX protocols, e.g. by shooting droplets of substrate-solution onto the chip-mounted microcrystals for reaction initiation, will enable recording snapshots of the reaction at short time-intervals. Alternatively, optically caged substrates can be used for reaction initiation. Additionally, I will make use of a recently established burst-series data collection mode, which enables the collection of many timepoints in rapid succession at a time-resolution of 1.35 ms. These short timeframes will make it possible to follow structural intermediates of the substrate during the catalysis. Finally, these experiments will be complemented by MD-simulations, aiming to connect the substeps of the conformational changes.