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

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a “dipped conformation” that has been previously proposed from molecular dynamics simulations. Taken together, the 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

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

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

   Institute for Biology, Leiden University, Sylviusweg 7, 333 BE Leiden

2. Teck Khiang Chua

   Cornell University

   Affiliation as printed

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

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

3. Madhur Srivastava

   Cornell University

   Affiliation as printed

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

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, New York 14853, United States

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, New York 1485, United States

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

   Institute for Biology, Leiden University, Sylviusweg 7, 333 BE Leiden

5. Zachary A. Maschmann

   Cornell University

   Affiliation as printed

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

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

6. Petr P. Borbat

   Cornell University

   Affiliation as printed

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

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, New York 14853, United States

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, New York 1485, United States

7. Jenna Chong

   Cornell University

   Affiliation as printed

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

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

8. Sheng Zhang

   Cornell University

   Affiliation as printed

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

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

9. Jack H. Freed

   Cornell University

   Affiliation as printed

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

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, New York 14853, United States

   Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853

   National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell University, Ithaca, New York 1485, United States

10. Ariane Briegel Aachen Institute for Biology, Leiden University

    Leiden University

    Affiliation as printed

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

    Institute for Biology, Leiden University, Sylviusweg 7, 333 BE Leiden

11. Brian R. Crane corresponding

    Cornell University

    Affiliation as printed

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

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 4853