Electrostatic interactions between rotor and stator in the bacterial flagellar motor

Abstract

Bacterial f lagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the f lagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.Many species of bacteria are propelled by flagella, each consisting of a thin helical propeller attached, via a flexible coupling, to a rotary motor in the cell membrane (for recent reviews, see refs. 1 and 2). The energy for rotation of the flagella comes from the transmembrane gradient of protons (3-5) or, in certain marine or alkaliphilic species, sodium ions (6). The molecular mechanism of torque generation is not understood. The proteins most closely involved are MotA, MotB, and FliG. MotA and MotB are integral membrane proteins (7-11) that function in proton conduction (12)(13)(14) and that are believed to form the stator, or nonrotating part, of the motor (9,(15)(16)(17). FliG is located on the cytoplasmic side of the membrane, and is thought to be a component of the rotor (18)(19)(20)(21)(22).In hypotheses for the mechanism of the flagellar motor, charged residues have often been suggested to play key roles, serving, for example, to regulate movements of the rotor or to control proton flow (5,(23)(24)(25). We recently identified charged residues essential for motor function in both the rotor protein FliG and the stator protein MotA in mutational analyses of these proteins (26,27). In FliG, the residues Arg-281, Asp-288, and Asp-289 are of primary importance for function, whereas residues Lys-264 and Arg-297 might have secondary roles [FliG residue numbers here are higher by two than those given in the previous study, owing to recent corrections to the Escherichia coli fliG sequence (28)]. In MotA, the charged residues most important for function are Arg-90 and Glu-98, whereas Glu-150 may have a secondary role. Mutant phenotypes suggest that charge is the most important feature of these residues and that the charged residues in each protein function redundantly. Single charge-neutralizing replacements had only mild effects on function, whereas double rep...

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

“…In contrast, in flagellar motor rotation, the rotor ring protein FliG, which determines the step size, is also responsible for switching rotational direction. The crystal structures of FliG show highly asymmetric nature (35,36), and the charged residues on the surface of its C-terminal domain are presumed to line up with their partner residues on the cytoplasmic domain of MotA for an electrostatic interaction between these two domains in a torque generation step (12). To achieve a symmetric elementary process of torque generation in both CCW and CW rotation, the C-terminal domain of FliG may need to go through a 180°rotation to reorient its charged residues in the two opposite directions.…”

“…FliF, FliG, FliM, and FliN form the rotor called the MS-C ring; FliF forms the MS ring, which spans through the cytoplasmic membrane with the M ring portion, and the remaining three proteins form the C ring on the cytoplasmic side of the MS ring (11). An electrostatic interaction between the cytoplasmic domain of MotA and the C-terminal domain of FliG is thought to be involved in torque generation (12). The protonation and deprotonation of a highly conserved aspartic acid of MotB is efficiently coupled with torque generation (13,14).…”

Asymmetry in the clockwise and counterclockwise rotation of the bacterial flagellar motor

“…In E. coli, a motor stator complex (anchored to the peptidoglycan layer) is composed of four copies of MotA and two copies of MotB, comprising two proton-conducting transmembrane channels (2). Protonation and subsequent deprotonation of MotB Asp-32 at the cytoplasmic end of either channel is thought to drive conformational changes that exert forces on the periphery of the rotor, via electrostatic interactions between MotA and the rotor protein FliG (17,18). Recently, dynamic models of the flagellar motor incorporating the power-stroke mechanism have been proposed (19)(20)(21)(22), which explain most features of the CCW torque-speed curve, including its concave shape.…”

A molecular brake, not a clutch, stops the Rhodobacter sphaeroides flagellar motor

“…The motor is powered by the cell's ion-motive force, usually maintained by electron transport (respiration/ photosynthesis). The flow of ions through peptidoglycan-bound stator complexes, down an electrochemical gradient into the cytoplasm, is responsible for rotor rotation via electrostatic interactions at the rotor-stator interface (4). Stator units can independently engage/disengage from the rotor (5), resulting in stepwise changes in speed (6).…”