ACERT Highlights: ACERT's Service and Collaborative Projects

National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

ACERT's Service and Collaborative Projects

Engineered Chemotaxis Core Signaling Units Studied by Pulse-Dipolar ESR Indicate A Constrained Kinase-Off State

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar electron-spin resonance spectroscopy (PDS) (cf. Fig.), as well as small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The PDS results recovered structures not seen by cryo-EM. The kinase-off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. These data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

Publication: Sci. Signal. 13, eabc1328 (2020). PMC7790435.

Fig. Spin-labeled ADP reports on the CheA ATP-binding pocket. Schematics (right) denote label positions in free CheA or the foldon complexes (red dots represent R1 nitroxide, blue dots represent ADP-NO). Baseline-corrected DEER time domain data (left) before (red) and after (black) wavelet denoising and resulting distance distributions (right, black) with error bounds (red) for (A) ADP-NO with free CheA; (B) ADP-NO, Tm CheA, CheW, and Tar foldon (TarFo); (C) ADP-NO with free CheA E588C; and (D) CheA E588C-R1 with ADP-NO, CheW, and TarFo.

Alise R. Muok (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, Institute for Biology, Leiden University, Sylviusweg, Leiden, Netherlands)
Teck Khiang Chua (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY)
Madhur Srivastava (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, ACERT)
Wen Yang (Institute for Biology, Leiden University, Sylviusweg, Leiden, Netherlands)
Zach Maschmann (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY)
Petr P. Borbat (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, ACERT)
Jenna Chong, Sheng Zhang (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY)
Jack H. Freed (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, ACERT)
Ariane Briegel (Institute for Biology, Leiden University, Sylviusweg, Leiden, Netherlands)
Brian R. Crane (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY,)

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

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National Institute of
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