Analysis of the Cytoplasmic Ring of the Bacterial Flagellar Motor

Biological motility comes in a variety of mechanisms, from the limbs of an elephant to the flagella of microorganisms. Bacterial and archeal flagella are powered by nanoscopic flagellar motors. The flagellar motor is a rotary nano-turbine that is powered by the influx of cations into the bacterium and coupled to the chemotaxis signaling system. In an attempt to further understand the structure and function of the flagellar motor, we have studied the mechanism of switching and the structure of the cytoplasmic torque ring.;Biochemical data show that when the chemotatic response regulator CheY is phosphorylated (CheY-P), it binds to FliM located in the cytoplasmic ring of the flagellar motor and signals the motor to switch direction of rotation. The switch in rotation is apparently achieved by CheY-P modulating the interaction between FliM and FliG. We have investigated the FliM/FliG interaction in response to CheY-P in order to study the mechanism of switching. Our experiments have shown that when CheY-P binds to FliM, it can displace the C-terminal domain of FliG, leaving the middle domain of FliG in contact with FliM. We propose the following mechanism of switching. During counter-clockwise rotation, both domains of FliG are in contact with FliM. The switch is achieved when CheY-P displaces the C-terminal domain of FliG from FliM causing the motor to rotate in the clockwise direction.;We then solved the crystal structure of the middle domain of FliM complexed with the middle and C-terminal domains of FliG. The structure showed only one contact surface between FliM and FliG and a dramatic rearrangement of the domains of FliG compared to previous structures. To verify this new compact form of FliG, we performed residual dipolar coupling analysis on the solution structure of FliG. The data suggests that FliG has a compact geometry in solution that supports the compact crystal structure. The complex structure was then fitted into the 3D cryo-electron tomography reconstructions of freeze-fractured flagellar motors. The fitting allowed for a model of the outer lobe of the cytoplasmic ring to be constructed. The ring is composed of 34 subunits of the complex, suggesting that in vivo FliM and FliG exist in a 1:1 stoichiometry.