Biology subjects

Eyles, S. J.

Publications and source records attributed to Eyles, S. J..

4 recordsLinked to original sources

bioRxiv2023

Proteomic survey of the DNA damage response in Caulobacter crescentus reveals evidence of post-transcriptional control

The bacterial DNA damage response is a critical, coordinated response to endogenous and exogenous sources of DNA damage. Response dynamics are dependent on coordinated synthesis and loss of relevant proteins. While much is known about its global transcriptional control, changes in protein abundance that occur upon DNA damage are less well characterized at the system level. Here, we perform a proteome-wide survey of the DNA damage response in Caulobacter crescentus. We find that while most protein abundance changes upon DNA damage are readily explained by changes in transcription, there are exceptions. The survey also allowed us to identify the novel DNA damage response factor, YaaA, which has been overlooked by previously published, transcription- focused studies. A similar survey in a {Delta}lon strain was performed to explore lons role in DNA damage survival. The {Delta}lon strain had a smaller dynamic range of protein abundance changes in general upon DNA damage compared to the wild type strain. This system-wide change to the dynamics of the response may explain this strains sensitivity to DNA damage. Our proteome survey of the DNA damage response provides additional insight into the complex regulation of stress response and nominates a novel response factor that was overlooked in prior studies. IMPORTANCEThe DNA damage response helps bacteria to react to and potentially survive DNA damage. The mutagenesis induced during this stress response contributes to the development of antibiotic resistance. Understanding how bacteria coordinate their response to DNA damage could help us to combat this growing threat to human health. While the transcriptional regulation of the bacterial DNA damage response has been characterized, this study is the first to our knowledge to assess the proteomic response to DNA damage in Caulobacter.

microbiology↗

bioRxiv2022

Bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of CheA

Motile bacteria have a chemotaxis system that enables them to sense their environment and direct their swimming towards favorable conditions. Chemotaxis involves a signaling process in which ligand binding to the extracellular domain of the chemoreceptor alters the activity of the histidine kinase, CheA, bound [~]300 [A] away to the distal cytoplasmic tip of the receptor, to initiate a phosphorylation cascade that controls flagellar rotation. The cytoplasmic domain of the receptor is thought to propagate this signal via changes in dynamics and/or stability, but it is unclear how these changes modulate the kinase activity of CheA. To address this question, we have used hydrogen deuterium exchange mass spectrometry to probe the structure and dynamics of CheA within functional signaling complexes of the E. coli aspartate receptor cytoplasmic fragment, CheA, and CheW. Our results reveal that stabilization of the P4 catalytic domain of CheA correlates with kinase activation. Furthermore, differences in activation of the kinase that occur during sensory adaptation depend on receptor destabilization of the P3 dimerization domain of CheA. Finally, hydrogen exchange properties of the P1 domain that bears the phosphorylated histidine identify the dimer interface of P1/P1 in the CheA dimer and support an ordered sequential binding mechanism of catalysis, in which dimeric P1/P1 has productive interactions with P4 only upon nucleotide binding. Thus stabilization/destabilization of domains is a key element of the mechanism of modulating CheA kinase activity in chemotaxis, and may play a role in the control of other kinases.

biochemistry↗

bioRxiv2021

A Cryptic K48 Ubiquitin Chain Binding Site on UCH37 is Required for its Role in Proteasomal Degradation

Degradation by the 26S proteasome is an intricately regulated process fine-tuned by the precise nature of ubiquitin modifications attached to a protein substrate. By debranching ubiquitin chains composed of K48 linkages, the proteasome-associated ubiquitin C-terminal hydrolase UCHL5/UCH37 serves as a positive regulator of protein degradation. How UCH37 achieves specificity for K48 chains is unclear. Here, we use a combination of hydrogen-deuterium mass spectrometry, chemical crosslinking, small-angle X-ray scattering, NMR, molecular docking, and targeted mutagenesis to uncover a cryptic K48 ubiquitin chain specific binding site on the opposite face of UCH37 relative to the canonical S1 ubiquitin-binding site. Biochemical assays demonstrate the K48 chain-specific binding site is required for chain debranching and proteasome-mediated degradation of proteins modified with branched chains. Using quantitative proteomics, translation shutoff experiments, and linkage-specific affinity tools, we then identify specific proteins whose degradation depends on the debranching activity of UCH37. Our findings suggest that UCH37 and potentially other DUBs could use more than one S1 site to perform different biochemical functions.

biochemistry↗

bioRxiv2019

Hydrogen exchange of chemoreceptors in functional complexes suggests protein stabilization mediates long-range allosteric coupling

Bacterial chemotaxis receptors form extended hexagonal arrays that integrate and amplify signals to control swimming behavior. Transmembrane signaling begins with a 2 [A] ligand-induced displacement of an alpha helix in the periplasmic and transmembrane domains, but it is not known how the cytoplasmic domain propagates the signal an additional 200 [A] to control the kinase CheA bound to the membrane-distal tip of the receptor. The receptor cytoplasmic domain has previously been shown to be highly dynamic as both a cytoplasmic fragment (CF) and within the intact chemoreceptor; modulation of its dynamics are thought to play a key role in signal propagation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) of functional complexes of CF, CheA, and CheW bound to vesicles in native-like arrays reveals that the CF is well-ordered only in its protein interaction region where it binds CheA and CheW. Rapid exchange is observed throughout the rest of the CF, with both uncorrelated (EX2) and correlated (EX1) exchange patterns, suggesting the receptor cytoplasmic domain retains disorder even within functional complexes. HDX rates are increased by inputs that favor the kinase-off state. We propose that chemoreceptors achieve long-range allosteric control of the kinase through a coupled equilibrium: CheA binding in a kinase-on conformation stabilizes the cytoplasmic domain, and signaling inputs that destabilize this domain (ligand binding and demethylation) disfavor CheA binding such that it loses key contacts and reverts to a kinase-off state. This study reveals the mechanistic role of an intrinsically disordered region of a transmembrane receptor in long-range allostery.

biochemistry↗