Architecture Of The Bacterial Flagellar Rotor Elucidated With X-Ray Crystallography And Pulsed Dipolar Esr Spectroscopy

Abstract

Bacteria swim through liquid media propelled by rotating flagella, driven by a highly complex motor at the base. In response to stimuli, the motor can reverse rotation allowing the bacteria to stop and change direction almost instantaneously. The rotor proteins FliG, FliM, and FliN form the switch complex to generate the cytoplasmic rotor or C-ring. The switch complex is essential for torque generation, binding the response regulator phosphorylated CheY, and switching. Some bacteria contain FliY, which comprises of a FliN homologue and an additional domain with conserved residues from the CheC phosphatase family. There are crystal structures for most of the major rotor components, but not for FliY or complexes containing FliY. To advance our understanding of motor switching, more comprehensive structural information on the switch complex proteins and their assembly state is needed. I determined the structure of FliY and characterized its biochemical properties. My studies show that FliY functions as an active phosphatase in Thermotoga maritima in spite of the presence of two other phosphatases in the chemotaxis signaling system. Interactions of FliY with other rotor proteins were studied. This information was used to develop a model of FliY arrangement in the switch complex. I also investigated the arrangement of FliM and FliG in the C-ring with X-ray crystallography and pulsed dipolar electron spin resonance spectroscopy (PDS). I determined the crystal structure of a FliG:FliM complex that produces an arc with a radius consistent with the C-ring. However, the antiparallel arrangement of subunits in the crystal structure does not represent the interaction of components in solution phase. PDS data shows that FliM and FliG interact through their middle domains in a parallel fashion. Higher order complexes are mediated by interactions between the C-terminal domain of FliG and the middle domain of a neighboring FliG. Such cross-dimer interactions help to polymerize FliG around the C-ring and could explain the high degree of cooperativity observed upon rotational switching. PDS studies report changes the middle domain of FliM undergoes in the presence of phosphorylated CheY. The crystal structures and spectroscopic studies reported herein provided new insights about the architecture of the C-ring.