Conformational change in the stator of the bacterial flagellar motor

MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct proton s across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rot ation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive mo vement of the rotor. To test for conformational changes, we examined the pr otease susceptibility of MotA in membrane-bound complexes with either wild- type MotB or MotB mutated at residue 32. Small, uncharged replacements of A sp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of pro teolytic fragments. The conformational change does not require any flagella r proteins besides MotA and MotB, as it was still seen in a strain that exp resses no other flagellar genes. It affects a cytoplasmic domain of MotA th at contains residues known to interact with the rotor, consistent with a ro le in the generation of torque. Influences of key residues of MotA on confo rmation were also examined. Pro173 of MotA, known to be important for rotat ion, is a significant determinant of conformation: Dominant Pro173 mutation s, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 a nd Glu98, residues of MotA that engage in electrostatic interactions with t he rotor, appear not to be strong determinants of conformation of the MotA/ MotB complex in membranes. We note sequence similarity between MotA and Exb B, a cytoplasmic-membrane protein that energizes outer-membrane transport i n Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.