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National Biomedical Center
for Advanced Electron Spin Resonance Technology

Our research is supported by a grant from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

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Assembly States within the Bacterial Flagellar Switch Complex as Elucidated by Pulse Dipolar ESR

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 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 (see below). 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 as measured by PDS fit a self-consistent model that agrees 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 from clockwise and counterclockwise. 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. The sensitive and stable performance of PDS at ACERT was instrumental for this sophisticated study conducted over a year-long period on multiple samples at varying conditions.

Funding: P41GM105321 (to J.H. Freed), R01GM604664 (to B.R. Crane), R15GM063514 (to C.J. Halkides).

Publication: R. Sircar, P.P. Borbat, M.J. Lynch, J. Bhatnagar, M. Beyersdorf, C.J. Halkides, J.H. Freed, and B.R. Crane. J. Mol. Biol. 427, 867-886 (2015) (PMC4323944).

(A) Schematic representation of the flagellar motor. The location of proteins FliG and FliM, studied here by PDS, is indicated with a green arrow. (B) The dipolar signal for singly spin-labeled residues in FliM increases upon the addition of longer constructs of FliG. (C) The models of FliG-FliM heterotetramer consistent with the distances obtained from the PDS data of panel B. (D) Fitting the experimental distances to Gaussians help to deduce an overall model of FliG-FliM association consistent with the PDS data. (E) PDS distances derived from FliG-FliM complexes involving four spin labels interacting within the heterotetramer.
R. Sircar (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York)
P.P. Borbat (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY; ACERT)
M.J. Lynch, J. Bhatnagar (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York)
M. Beyersdorf, C.J. Halkides (Department of Chemistry and Biochemistry, Unversity of North Carolina Wilmington, Wilmington, NC)
J.H. Freed (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York; ACERT)
and B.R. Crane (Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York)
June, 2016