Room-temperature crystallography reveals altered binding of small-molecule fragments to PTP1B

Abstract

Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy*, Hill*, 2018). Here we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly -- but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryogenic-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

“…These differences between room-temperature and cryogenic crystallography structures have been experimentally observed for several proteins. [86][87][88][89][90][91][92][93] A careful analysis of our NQO1 structure has further revealed that residues Tyr128 and Phe232, which play a key role in the function of the protein, [94][95][96][97][98] show an unexpected flexibility within the crystals (Fig. 7(a)).…”

Perspective: Structure determination of protein-ligand complexes at room temperature using X-ray diffraction approaches

“…These tools will be highly applicable to work at ambient temperature and we envisage combined studies using both crystals under cryogenic conditions and at ambient temperature ( Krojer et al, 2020 ). While several studies report ligand binding at room temperature and 100 K ( Maeki et al, 2020 ; Mehlman et al, 2022 ), a full understanding of which protein-ligand interactions are likely to display such differences remains elusive. Similarly benefiting from computational power are docking software and machine learning (ML) algorithms for ligands and drug molecules identification and/or generation.…”

“…For example, it is recognised that data collection under the cryogenic conditions (100 K) that are standard in X-ray crystallography may result in binding modes or interactions that are not fully consistent with those occurring under physiological conditions ( Helliwell, 2020 ). A fascinating example was recently provided by a systematic comparison of the binding of fragments to the enzyme protein tyrosine phosphatase, PTP1B ( Mehlman et al, 2022 ). Here, fragments were soaked into crystals that were then either cryo-cooled conventionally, harvested for room temperature data collection in loops or measured in situ within crystallisation plates.…”

“…Here, fragments were soaked into crystals that were then either cryo-cooled conventionally, harvested for room temperature data collection in loops or measured in situ within crystallisation plates. Intriguingly, significant differences were observed for some fragments between the 100 K structures and those at room temperature despite the original soaking and presumably binding being identical ( Mehlman et al, 2022 ). In this study, different temperatures revealed distinct binding poses and protein-ligand interactions, novel binding sites and changes in water structure together with a previously uncharacterised allosteric binding site.…”

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM

“…This automation in modeling is needed especially in light of advances in data collection automation and fast detectors. These tools have revolutionized the field of X-ray crystallography, enabling high-temperature datasets, time-resolved experiments, and high-throughput data collection [46][47][48][49][50] . With the ability to capture different conformations, there is a growing demand for methods that can detect protein alternative conformers to extract as much biological information as possible.…”