Biology subjects

Wagstaff, J. M.

Publications and source records attributed to Wagstaff, J. M..

5 recordsLinked to original sources

bioRxiv2025

Targeting a Pleckstrin Homology Domain with a Lysine-Reactive Cova-lent Binder

Brutons Tyrosine Kinase (BTK) is a validated target for haematological malignancies, with numerous FDA approved inhibitors on the market. Current therapies target the highly conserved ATP binding site and hence limit the therapeutic index given the sites highly conserved nature across the kinome. We explore a novel approach for BTK inhibition, by targeting the PH domain-mediated membrane recruitment and activation of BTK. We have identified a fragment which covalently labels a lysine in the inositol phosphate (PIP3) binding site. Fragment growth and an extensive structure-binding relationship study uncovered 27 crystal structures and a best-in-class analog, 24. Evaluation of pKa values of the targeted lysine in BTK and other PH domains suggests this as a more general approach to PH domain inhibition.

biochemistry↗

bioRxiv2024

Discovery and chemical optimisation of a Potent, Bi-cyclic (Bicycle(R)) Antimicrobial Inhibitor of Escherichia coli PBP3

Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of {beta}-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of novel, bicyclic peptide (Bicycle(R)) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent a novel non-{beta}-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors.

biochemistry↗

bioRxiv2022

Cytomotive actins and tubulins share a polymerisation switch mechanism conferring robust dynamics

Protein filaments are used in myriads of ways to organise other molecules in space and time within cells. Some filament-forming proteins couple the hydrolysis of nucleotides to their polymerisation cycle, thus powering the directed movement of other molecules. These filaments are termed cytomotive. Only members of the actin and tubulin protein superfamilies are known to form cytomotive filaments. We sought to examine the basis of cytomotivity via structural studies of the polymerisation cycles of actin and tubulin homologues from across the tree of life. We analysed published data and performed new structural experiments designed to disentangle functional components of these complex filament systems. In sum, our analysis demonstrates the existence of shared subunit polymerisation switches amongst both cytomotive actins and tubulins, i.e. the conformation of subunits switches upon assembly into filaments. Such cytomotive switches explain filament robustness, by enabling the coupling of kinetic and structural polarities required for useful cytomotive behaviours, and by ensuring that single cytomotive filaments do not fall apart.

molecular biology↗

bioRxiv2019

Bactofilins form non-polar filaments that bind to membranes directly

Bactofilins are small beta-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions: filaments in Caulobacter crescentus are involved in cell stalk formation whereas Myxococcus xanthus filaments aid chromosome segregation and motility. The precise molecular architecture of bactofilin filaments has remained unclear. Here we revealed by sequence analyses and electron microscopy that in addition to wide distribution across bacteria and archaea, bactofilins are also present in a few eukaryotic cells such as oomycetes. The sole bactofilin from Thermus thermophilus was demonstrated to form constitutive filaments and cryo-EM analysis revealed that protofilaments formed through end-to-end association of the beta-helical domains. Using a nanobody against Thermus bactofilin we determined the near-atomic filament structure, showing that the filaments are non-polar, with subunits arranged head-to-head and tail-to-tail. A polymerisation-impaired mutant F105R, that disrupts one of the two protofilament interfaces, enabled crystallisation. The crystal structure also revealed non-polar protofilaments, and the dominance of the beta-stacking interface that formed despite the inhibiting mutation. To confirm the generality of the lack of polarity, we performed co-evolutionary analysis of a large set of sequences. Finally, using Thermus bactofilin, we determined that the N-terminal disordered tail of the protein is responsible for direct binding to lipid membranes both on liposomes and by electron cryotomography in E. coli cells. The tail is conserved, suggesting that membrane binding is likely a general feature of these very common but only recently discovered filaments of the prokaryotic cytoskeleton.

biochemistry↗

bioRxiv2016

A polymerisation-associated conformational switch in FtsZ that enables treadmilling

Bacterial cell division in many organisms involves a constricting cytokinetic ring that is orchestrated by the tubulin-like protein FtsZ. FtsZ forms dynamic filaments close to the membrane at the site of division that have recently been shown to treadmill around the division ring, guiding septal wall synthesis.\n\nHere, using X-ray crystallography of Staphylococcus aureus SaFtsZ we reveal how an FtsZ can adopt two functionally distinct conformations, open and closed. The open form is found in SaFtsZ filaments formed in crystals and also in soluble filaments of E. coli FtsZ as deduced by cryoEM. The closed form is found within several crystal forms of two non-polymerising SaFtsZ mutants and corresponds to many previous FtsZ structures from other organisms.\n\nWe argue that FtsZ undergoes a polymerisation-associated conformational switch. We show that such a switch provides explanations for both how treadmilling may occur within a single-stranded filament, and why filament assembly is cooperative.

microbiology↗