Biology subjects

Bradshaw, N.

Publications and source records attributed to Bradshaw, N..

6 recordsLinked to original sources

bioRxiv2025

SpoIIE drives asymmetric cell division in B. subtilis by sequential modulation of the cytokinesis machinery

To form a dormant spore, Bacillus subtilis and related endospore-forming bacteria divide asymmetrically to generate daughter cells of unequal size, the smaller of which becomes the spore. The transmembrane protein SpoIIE repositions the cell division machinery and controls cytokinesis during sporulation, but the molecular basis for the precise placement of the asymmetrical division site to the quarter cell point is unknown. Here, we applied live-cell fluorescence microscopy techniques to reveal that SpoIIE localizes with the treadmilling components of the cell division machinery. We found that SpoIIE opposes the inhibitory activity of the MinCD complex, which prevents assembly of Z-rings near the cell poles. Cells expressing a variant of SpoIIE with its transmembrane region replaced by an unrelated transmembrane anchor assembled condensed Z-rings that were unable to initiate constriction. This reveals a new function of SpoIIE and a possible checkpoint licensing cytokinesis downstream of Z-ring condensation. Potentially explaining the role of SpoIIE in cytokinesis, we demonstrated that SpoIIEs transmembrane region interacts with DivIB, an enigmatic structural component of the cell wall synthesis complex required for cytokinesis during sporulation. Finally, we found that FtsZ filaments are unusually short during sporulation, which requires the transmembrane domain of SpoIIE. Together, these results demonstrate that SpoIIE sequentially influences polar divisome assembly at distinct steps to drive asymmetric cell division.

microbiology↗

bioRxiv2024

Dual-Action Kinase Inhibitors Control p38α MAP Kinase Threonine Dephosphorylation

Reversible protein phosphorylation directs essential cellular processes including cell division, cell growth, cell death, inflammation, and differentiation. Because protein phosphorylation drives diverse diseases, kinases and phosphatases have been targets for drug discovery, with some achieving remarkable clinical success. Most protein kinases are activated by phosphorylation of their activation loops, which shifts the conformational equilibrium of the kinase towards the active state. To turn off the kinase, protein phosphatases dephosphorylate these sites, but how the conformation of the dynamic activation loop contributes to dephosphorylation was not known. To answer this, we modulated the activation loop conformational equilibrium of human p38 MAP kinase with existing kinase inhibitors that bind and stabilize specific inactive activation loop conformations. From this, we discovered three inhibitors that increase the rate of dephosphorylation of the activation loop phospho-threonine by the PPM serine/threonine phosphatase WIP1. Hence, these compounds are "dual-action" inhibitors that simultaneously block the active site and stimulate p38 dephosphorylation. Our X-ray crystal structures of phosphorylated p38 bound to the dual-action inhibitors reveal a shared flipped conformation of the activation loop with a fully accessible phospho-threonine. In contrast, our X-ray crystal structure of phosphorylated apo human p38 reveals a different activation loop conformation with an inaccessible phospho-threonine, thereby explaining the increased rate of dephosphorylation upon inhibitor binding. These findings reveal a conformational preference of phosphatases for their targets and suggest a new approach to achieving improved potency and specificity for therapeutic kinase inhibitors.

biochemistry↗

bioRxiv2024

Docking interactions determine substrate specificity of members of a widespread family of protein phosphatases

How protein phosphatases achieve specificity for their substrates is a major outstanding question. PPM family serine/threonine phosphatases are widespread in bacteria and eukaryotes, where they dephosphorylate target proteins with a high degree of specificity. In bacteria, PPM phosphatases control diverse transcriptional responses by dephosphorylating anti-anti-sigma factors of the STAS domain family, exemplified by B. subtilis phosphatases SpoIIE, which controls cell-fate during endospore formation, and RsbU, which initiates the General Stress Response. Using a combination of forward genetics, biochemical reconstitution, and AlphaFold2 structure prediction, we identified a conserved, tripartite substrate docking interface comprised of three variable loops on the surface of the PPM phosphatase domain that recognize the three-dimensional structure of the substrate protein. Non-conserved amino acids in these loops facilitate the accommodation of the cognate substrate and prevent dephosphorylation of the non-cognate substrate. Together, single-amino acid substitutions in these three elements cause an over five-hundred fold change in specificity. Our data additionally suggest that substrate-docking interactions regulate phosphatase specificity through a conserved allosteric switch element that controls the catalytic efficiency of the phosphatase by positioning the metal cofactor and substrate. We hypothesize that this is a generalizable mechanistic model for PPM family phosphatase substrate specificity. Importantly, the substrate docking interface with the phosphatase is only partially overlapping with the much more extensive interface with the upstream kinase, suggesting the possibility that kinase and phosphatase specificity evolved independently.

biochemistry↗

bioRxiv2024

A General Mechanism for the General Stress Response in Bacteria

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

microbiology↗

bioRxiv2022

From primordial clocks to circadian oscillators

Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 [A]. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.

biochemistry↗

bioRxiv2019

A conserved regulatory switch controls phosphatase activity and specificity

Protein phosphatases must be regulated and specific for their substrates; how this control is achieved is critical for signaling by reversible phosphorylation. We recently reported the discovery of a regulatory switch that controls the activity of the PP2C family serine/threonine phosphatase SpoIIE from the bacterium Bacillus subtilis. This regulatory switch activates SpoIIE during spore development by forming the catalytically essential metal-binding site that is conserved across the PP2C family. We hypothesized that this switch is a conserved platform for regulating other PP2C phosphatases. An orthologous phosphatase from B. subtilis, RsbU, is activated under stress conditions and responds to different signals and acts on a different phosphoprotein substrate than SpoIIE. Using a combination of biochemical and genetic approaches, we find that broad features of the regulatory mechanism are conserved between SpoIIE and RsbU but that each phosphatase has adapted its response to be most appropriate for the distinct biological outputs it controls. In both cases, the switch accomplishes this by integrating substrate binding and recognition with regulatory inputs to control metal cofactor binding and catalysis. Thus, the switch is a conserved and mechanistically flexible regulatory platform that controls phosphatase activity and substrate specificity.

biochemistry↗