Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography
Abstract
β-Lactamases hydrolyse β-lactam antibiotics and are key antimicrobial resistance determinants in globally important bacterial pathogens. Here, we report on the use of time-resolved room-temperature drop-on-fixed-target serial crystallography, complemented by classical single-crystal crystallography and molecular simulations, to investigate reversible covalent inhibition of the serine β-lactamase CTX-M-15 by the diazabicyclooctane inhibitor avibactam, used to treat infections by β-lactamase producing organisms. A covalent ring-opened avibactam carbamoyl complex is observed in crystal structures 80 ms after mixing, which rearranges on a 1.3 s to 600 s time scale. Together with molecular dynamics simulations and density functional theory calculations based on our experimental structures, our data suggest that in the initially observed species the avibactam N-sulfate nitrogen is poorly positioned to attack the carbamoyl carbon to initiate the 5-exo-trig recyclisation required for inhibitor reformation. This complex then undergoes conformational changes that potentially prime it for this intramolecular reaction. These data reveal the conformational dynamics and stereoelectronic basis of diazobicyclooctane inhibition and demonstrate how the combination of room-temperature serial crystallography and molecular simulations can capture ligand motions in enzyme active sites on millisecond-to-second timescales, delivering mechanistically relevant understanding of the dynamic behaviour of enzyme-bound species.
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BibTeX
@article{hinchliffe2026timeresolved,
title = {{Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography}},
author = {Philip Hinchliffe and Catherine L. Tooke and Michael Beer and Pierre Aller and Jos J. A. G. Kamps and Laura Parkinson and Tiankun Zhou and Nicholas Devenish and Do-Heon Gu and Anastasya Shilova and Matthew J. Rodrigues and Abbey Telfer and Agata Butryn and Marko Hanževački and Emily I. Freeman and Sehan Park and Christopher J. Schofield and Jaehyun Park and Robin L. Owen and Adrian J. Mulholland and Allen M. Orville and James Spencer},
journal = {Communications Chemistry},
year = {2026},
month = nov,
doi = {10.1038/s42004-026-02262-6},
url = {https://www.nature.com/articles/s42004-026-02262-6},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography
AU - Philip Hinchliffe
AU - Catherine L. Tooke
AU - Michael Beer
AU - Pierre Aller
AU - Jos J. A. G. Kamps
AU - Laura Parkinson
AU - Tiankun Zhou
AU - Nicholas Devenish
AU - Do-Heon Gu
AU - Anastasya Shilova
AU - Matthew J. Rodrigues
AU - Abbey Telfer
AU - Agata Butryn
AU - Marko Hanževački
AU - Emily I. Freeman
AU - Sehan Park
AU - Christopher J. Schofield
AU - Jaehyun Park
AU - Robin L. Owen
AU - Adrian J. Mulholland
AU - Allen M. Orville
AU - James Spencer
PY - 2026
DA - 2026/11/04
JO - Communications Chemistry
DO - 10.1038/s42004-026-02262-6
UR - https://www.nature.com/articles/s42004-026-02262-6
AB - β-Lactamases hydrolyse β-lactam antibiotics and are key antimicrobial resistance determinants in globally important bacterial pathogens. Here, we report on the use of time-resolved room-temperature drop-on-fixed-target serial crystallography, complemented by classical single-crystal crystallography and molecular simulations, to investigate reversible covalent inhibition of the serine β-lactamase CTX-M-15 by the diazabicyclooctane inhibitor avibactam, used to treat infections by β-lactamase producing organisms. A covalent ring-opened avibactam carbamoyl complex is observed in crystal structures 80 ms after mixing, which rearranges on a 1.3 s to 600 s time scale. Together with molecular dynamics simulations and density functional theory calculations based on our experimental structures, our data suggest that in the initially observed species the avibactam N-sulfate nitrogen is poorly positioned to attack the carbamoyl carbon to initiate the 5-exo-trig recyclisation required for inhibitor reformation. This complex then undergoes conformational changes that potentially prime it for this intramolecular reaction. These data reveal the conformational dynamics and stereoelectronic basis of diazobicyclooctane inhibition and demonstrate how the combination of room-temperature serial crystallography and molecular simulations can capture ligand motions in enzyme active sites on millisecond-to-second timescales, delivering mechanistically relevant understanding of the dynamic behaviour of enzyme-bound species.
SN - 2399-3669
ER -