A biochemical and physiological study of the stator complex of the bacterial flagellar motor

The bacterial flagellar motor derives its energy from the transmembrane proton (or in some species Na+-ion) gradient. The MotA and MotB integral membrane proteins together function to conduct protons across the membrane. The manner in which these proteins couple proton flow to motor rotation is not known. This study provides evidence for an interaction between MotA and MotB and presents initial investigations into their association state. Physiological characterization of MotA mutants provides evidence that two conserved Proline residues (positions 173 & 222 in E. coli ) function together with Asp32 of MotB in a site that serves to couple proton flow to motor rotation. Asp32 was identified previously as a likely binding site for protons within the MotA/B channel. A model for the Pro173/222, Asp32 site is proposed.;Results of a disulfide scanning study of the membrane spanning segment of MotB show that this region of the protein is alpha-helical and associates as a symmetrical dimer. The position of Asp32 residues in the MotB dimer suggests that there are two proton channels per MotA/MotB complex. Experiments testing the accessibility of introduced cysteines in MotB to membrane permeable and nonpermeable Cysteine selective reagents shows that at least two positions near the middle of the membrane segment (positions 39 & 42) are accessible to stable, polar reagents, probably because they face the lumen of an aqueous pore. Further cross-linking studies provide biochemical evidence for the proximity of residue 173 in MotA to residue 32 of MotB and suggest that the complex contains multiple copies of MotA which are differentially labile to detergent.