Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Science Signaling, vol. 13

Abstract

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), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. 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. Together, 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.

Authors 11

1. Alise R. Muok Aachen Institute for Biology, Leiden University

   Leiden University · Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

   Institute for Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, Netherlands

2. Teck Khiang Chua

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

3. Madhur Srivastava

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, NY 14853, USA

4. Wen Jing Yang Aachen Institute for Biology, Leiden University

   Leiden University

   Affiliation as printed

   Institute for Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, Netherlands

5. Zachary A. Maschmann

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

6. Petr P. Borbat

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, NY 14853, USA

7. Jenna Chong

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

8. Sheng Zhang

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

9. Jack H. Freed

   Cornell University

   Affiliation as printed

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, NY 14853, USA

10. Ariane Briegel Aachen Institute for Biology, Leiden University

    Leiden University

    Affiliation as printed

    Institute for Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, Netherlands

11. Brian R. Crane corresponding

    Cornell University

    Affiliation as printed

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA