Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography

Abstract

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.

Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket

“…Finally, although crystal-packing-related artefacts can be minimized by co-crystallization, these results highlight the importance of exploring binding-site flexibility and ligand mobility during structure-guided drug discovery. Roomtemperature data collection has been used to probe different active-site conformations and to avoid cryogenic cooling artefacts (Huang et al, 2022).…”

SDU – software for high-throughput automated data collection at the Swiss Light Source

“…In the past few years MX experiment methodology has experienced clear branching. On the one hand, beamline staff focused on development and support of more sophisticated types of experiments, such as serial crystallography, pumpprobe, temperature gradient, and new sample delivery modes, namely jet, fixed target and levitation (Oghbaey et al, 2016;Martiel et al, 2019;Huang et al, 2022;Kepa et al, 2022;Pearson & Mehrabi, 2020;Keedy et al, 2015). On the other, standardization and high throughput of standard rotational and the more mature fixed target experiments have become the holy grail of many MX beamlines.…”

Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi

“…The importance of determining ambient (room) temperature (RT) and even above RT crystal structures to understand protein function, allostery and the binding of ligands or drugs is becoming ever more recognized (Helliwell, 2020;Fraser et al, 2011;Fischer, 2021). Recent work has shown differences in the response of conformational states to radiation damage between RT and 100 K (Yabukarski et al, 2022), and in allosteric networks (Keedy et al, 2018) and ligand binding (Huang et al, 2022). Significant challenges remain in obtaining such structures without excessive radiation damage or when handling very small or fragile crystals, such as those of membrane proteins, large complexes or viruses.…”