Biology subjects

Thompson, L. K.

Publications and source records attributed to Thompson, L. K..

5 recordsLinked to original sources

bioRxiv2025

Selective Detection of a Key Region in Chemotaxis Signaling Protein Complexes by Solid-State NMR

Understanding how large protein complexes function requires tools that can resolve both structure and dynamics in their native assembly states. Here, we apply solid-state NMR (SSNMR) to selectively detect rigid regions of a receptor protein fragment in the context of the >500 kDa chemoreceptor signaling complex found in chemotactic bacteria. These complexes assemble into hexagonal arrays that network multiple active units together. The cytoplasmic fragment of the E. coli aspartate chemoreceptor exhibits dynamics on multiple timescales across different regions of the protein, and these dynamics differ between signaling states. We apply 13C-15N dipolar coupling-based SSNMR experiments to selectively probe the rigid portion (motions slower than millisecond timescale) of this protein, in the context of the full array structure. We optimized assembly methods to form native-like, homogeneous complexes capable of maintaining activity and sample integrity during extended NMR experiments with low electric-field NMR probe designs. A subset of the protein, approximately 100 residues at the membrane-distal tip of the chemoreceptor where it interacts with its associated kinase, was detected and identified as the most rigid region. Chemical shift changes for many residues in this rigid region were observed between NMR spectra of the kinase-on and kinase-off signaling states. This suggests conformational changes occur at the chemoreceptor tip during signaling, which have not been observed in previous studies of this system. These findings demonstrate a dynamics-based NMR spectral editing approach to selectively examine a key region of a large signaling protein within its macromolecular assembly. Statement of SignificanceLarge protein complexes are challenging to study, but understanding their mechanism is vital to gaining insight into many important biological processes. In this work we demonstrate the use of solid-state NMR to study such a system, bacterial chemotaxis receptor signaling complexes. We identified an in vitro assembly method that creates reproducible and durable complexes that retain activity for weeks of data collection. Selective NMR detection of a key region of one protein reveals significant differences between signaling states, indicating there are changes in conformation and dynamics that were not previously seen by cryo-electron tomography and crystallography of this system. This highlights the potential of solid-state NMR as a key tool in mechanistic studies of multi-protein complexes.

biochemistry↗

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↗

bioRxiv2022

Coupling comprehensive pesticide-wide association study to iPSC dopaminergic screening identifies and classifies Parkinson-relevant pesticides

Parkinsons disease (PD) is a complex, multi-factorial neurodegenerative disease, known to involve genetic, aging-related components, but also to be highly sensitive to environmental factors. In particular, ample evidence links pesticides to PD etiology. Here, establishing a field-to-bench paradigm, we have combined record-based exposure assessment in a population-based epidemiologic study of PD with testing in dopaminergic neurons produced from iPSCs to further identify and classify PD-relevant pesticides. First, agricultural pesticide-application records in California enabled us to investigate exposure to nearly 300 specific pesticides and PD risk in a comprehensive, pesticide-wide association study (PWAS). We implicated long-term exposure to 53 pesticide active ingredients in PD risk and identified their relevant co-exposure profiles. Second, to identify which of these pesticides might contribute to PD through direct effects on dopaminergic neurons, we employed a live-cell imaging screening paradigm in which neurons, definitively identified with a tyrosine hydroxylase reporter, were exposed to 43 of the high-risk pesticides. Using detailed morphometric measures, we found 10 pesticides were directly toxic to these neurons. Further, we analyzed pesticides typically used in combinations in cotton farming. Among these "cotton cluster" pesticides, co-exposures resulted in markedly greater toxicity than any single pesticide. Trifluralin was a pivotal driver of toxicity to dopaminergic neurons and led to marked mitochondrial dysfunction. Our field-to-bench paradigm may prove useful to mechanistically dissect pesticide exposure implicated in PD risk, and guide agricultural policy in the future.

neuroscience↗

bioRxiv2020

Strategies for identifying dynamic regions in protein complexes: flexibility changes accompany methylation in chemotaxis receptor signaling states

Bacterial chemoreceptors are organized in arrays composed of helical receptors arranged as trimers of dimers, coupled to a histidine kinase CheA and a coupling protein CheW. Ligand binding to the external domain inhibits the kinase activity, leading to a change in the swimming behavior. Adaptation to an ongoing stimulus involves reversible methylation and demethylation of specific glutamate residues. However, the exact mechanism of signal propagation through the helical receptor to the histidine kinase remains elusive. Dynamics of the receptor cytoplasmic domain is thought to play an important role in the signal transduction, and current models propose inverse dynamic changes in different regions of the receptor. We hypothesize that the adaptational modification (methylation) controls the dynamics by stabilizing a partially ordered domain, which in turn modulates the binding of the kinase, CheA. We investigated the difference in dynamics between the methylated and unmethylated states of the chemoreceptor using solid-state NMR. The unmethylated receptor (CF4E) shows increased flexibility relative to the methylation mimic (CF4Q). Methylation helix 1 (MH1) has been shown to be flexible in the methylated receptor. Our analysis indicates that in addition to MH1, methylation helix 2 also becomes flexible in the unmethylated receptor. In addition, we have demonstrated that both states of the receptor have a rigid region and segments with intermediate dynamics. The strategies used in the study for identifying dynamic regions are applicable to a broad class of proteins and protein complexes with intrinsic disorder and dynamics spanning multiple timescales. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/974428v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8a8b0forg.highwire.dtl.DTLVardef@1afb646org.highwire.dtl.DTLVardef@e7dbaaorg.highwire.dtl.DTLVardef@685c0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReceptors exhibit greater ns timescale dynamics in unmethylated vs methylated state C_LIO_LIMethylation helix 2 likely involved in increased flexibility of unmethylated state C_LIO_LIDynamics occur on multiple timescales in both states of the receptor C_LI

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↗