title: Assembly states of FliM and FliG within the flagellar switch complex.
abstract: At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS). A new crystal structure of an 11-unit FliG:FliM complex produces a large arc with a curvature consistent with the dimensions of the C-ring. Previously determined structures along with this new structure provided a basis to test switch complex assembly models. PDS combined with mutational studies and targeted cross-linking reveal that FliM and FliG interact through their middle domains to form both parallel and antiparallel arrangements in solution. Residue substitutions at predicted interfaces disrupt higher-order complexes that are primarily mediated by contacts between the C-terminal domain of FliG and the middle domain of a neighboring FliG molecule. Spin separations among multi-labeled components fit a self-consistent model that agree well with electron microscopy images of the C-ring. An activated form of the response regulator CheY destabilizes the parallel arrangement of FliM molecules to perturb FliG alignment in a process that may reflect the onset of rotation switching. These data suggest a model of C-ring assembly in which intermolecular contacts among FliG domains provide a template for FliM assembly and cooperative transitions.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Ria
authors.initials: R
authors.lastname: Sircar
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; National Biomedical Center for Advanced ESR Technology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Peter P
authors.initials: PP
authors.lastname: Borbat
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lynch
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Jaya
authors.initials: J
authors.lastname: Bhatnagar
authors.affiliation: Department of Chemistry and Biochemistry, Unversity of North Carolina Wilmington, Wilmington, NC 28403, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Beyersdorf
authors.affiliation: Department of Chemistry and Biochemistry, Unversity of North Carolina Wilmington, Wilmington, NC 28403, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Halkides
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; National Biomedical Center for Advanced ESR Technology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Jack H
authors.initials: JH
authors.lastname: Freed
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA. Electronic address: bc69@cornell.edu.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Crane
mesh_terms: D001426:Bacterial Proteins / Q000648:ultrastructure*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D005407:Flagella; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000648:ultrastructure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D020124:Thermotoga maritima / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C077389:FliN protein, Bacteria; C075660:Flig protein, Bacteria; D046912:Multiprotein Complexes; D013113:Spin Labels; C066821:FliM protein, Bacteria
keywords: X-ray crystallography; chemotaxis; flagella motor; protein–protein interactions; signal transduction