Engineered chemotaxis core signaling units indicate a constrained kinase-off state
Engineered chemotaxis core signaling units indicate a constrained kinase-off state
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.
Zhang Sheng、Muok Alise R.、Srivastava Madhur、Briegel Ariane、Crane Brian R.、Chua Teck Khiang、Yang Wen、Borbat Petr P.、Freed Jack H.、Maschmann Zach、Chong Jenna
Department of Chemistry and Chemical Biology, Cornell UniversityDepartment of Chemistry and Chemical Biology, Cornell University||Institute for Biology, Leiden UniversityDepartment of Chemistry and Chemical Biology, Cornell University||National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell UniversityInstitute for Biology, Leiden UniversityDepartment of Chemistry and Chemical Biology, Cornell UniversityDepartment of Chemistry and Chemical Biology, Cornell UniversityInstitute for Biology, Leiden UniversityDepartment of Chemistry and Chemical Biology, Cornell University||National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell UniversityDepartment of Chemistry and Chemical Biology, Cornell University||National Biomedical Center for Advanced ESR Technologies (ACERT), Cornell UniversityDepartment of Chemistry and Chemical Biology, Cornell UniversityDepartment of Chemistry and Chemical Biology, Cornell University
生物科学研究方法、生物科学研究技术分子生物学生物化学
Zhang Sheng,Muok Alise R.,Srivastava Madhur,Briegel Ariane,Crane Brian R.,Chua Teck Khiang,Yang Wen,Borbat Petr P.,Freed Jack H.,Maschmann Zach,Chong Jenna.Engineered chemotaxis core signaling units indicate a constrained kinase-off state[EB/OL].(2025-03-28)[2025-05-06].https://www.biorxiv.org/content/10.1101/2020.03.27.011866.点此复制
评论