Biology subjects
Publications and source records attributed to Pearce, N. M..
In their recent commentary in Protein Science, Jaskolski et al. analyze three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, claim that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.
Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.
Tetraspanins are ubiquitous eukaryotic membrane proteins that contribute to a variety of signaling processes by spatially organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into so-called tetraspanin-enriched microdomains is unknown. Here we present crystal structures of the large extracellular loop of CD9 in complex with nanobodies 4C8 and 4E8; and, the cryo-EM structure of 4C8-bound CD9 in complex with its prototypical partner EWI-F. The CD9 - EWI-F complex displays a tetrameric arrangement with two centrally positioned EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F single-pass transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at the C and D loop, in agreement with 4C8 binding at the ends of the CD9 - EWI-F cryo-EM complex. Overall, the 4C8 - CD9 - EWI-F - EWI-F - CD9 - 4C8 complexes varied from nearly two-fold symmetric (i.e. with the two CD9 - 4C8 copies in nearly anti-parallel orientation) to ca. 50{degrees} bent arrangements. Since membrane helices h1 and h2 and the EC2 D-loop have been previously identified as sites for tetraspanin homo-dimerization, the observed linear but flexible arrangement of CD9 - EWI-F with potential CD9 - CD9 homo-dimerization at either end provides a new concatenation model for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.
Allostery is an inherent feature of proteins and provides alternative routes to regulating function. Small-molecule allosteric inhibitors are often desirable; however, it remains challenging to identify surface sites in proteins which can bind small molecules and modulate function. We identified new allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal \"hidden\" low-occupancy conformational states for protein and ligands. Our results converge on a new allosteric site that is conformationally coupled to the active-site WPD loop, a hotspot for fragment binding, not conserved in the closest homolog, and distinct from other recently reported allosteric sites in PTP1B. Targeting this site with covalently tethered molecules allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure revealed by multitemperature crystallography can be exploited for developing allosteric modulators.
SynopsisA high-throughput method is described for crystal soaking using acoustic droplet ejection, and its effectiveness demonstrated.\n\nAbstractBright light sources, agile robotics, and fast detectors are continually reducing the time it takes to perform an X-ray diffraction experiment, making high throughput experiments more feasible than ever. But this is also pushing the upstream bottleneck towards sample preparation, even for robust and well characterised crystal systems. Crystal soaking is routinely used to generate protein-ligand complex structures, yet protein crystals are often sensitive to changes in solvent composition, and frequently require gentle or careful stepwise soaking techniques, limiting overall throughput. Here, we describe the use of acoustic droplet ejection for soaking of protein crystals with small molecules, and show that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 minutes. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables targeted placement of the compound/solvent away from crystals and towards drop edges, allowing for gradual diffusion of solvent across the drop. This ensures both an improvement in reproducibility of X-ray diffraction and an increased solvent tolerance of the crystals, thus enabling higher effective compound soaking concentrations. We detail the technique here with examples from the protein target JMJD2D, a histone lysine demethylase, having roles in cancer and the focus of active structure based drug design efforts.