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

• Applicant: Dr. Eike Schulz
• Subject Area: Structural Biology ; Biochemistry ; Biophysics
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458246365

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.

• DFG Programme: Research Grants
• International Connection: Denmark
• Cooperation Partner: Professor Dr. Kresten Lindorff-Larsen, Ph.D.