Membrane Segment Organization in the Stator Complex of the Flagellar Motor: Implications for Proton Flow and Proton-Induced Conformational Change

Abstract

MotA and MotB are membrane proteins that form the stator of the bacterial flagellar motor. Each motor contains several MotA 4 MotB 2 complexes, which function independently to conduct protons across the membrane and couple proton flow to rotation. The mechanism of rotation is not understood in detail but is thought to involve conformational changes in the stator complexes driven by proton association/dissociation at a critical Asp residue of MotB (Asp 32 in the protein of Escherichia coli). MotA has four membrane segments and MotB has one. Previous studies using targeted disulfide crosslinking showed that the membrane segments of the two MotB subunits are together at the center of the complex, surrounded by the TM3 and TM4 segments of the four MotA subunits. Here, the cross-linking studies are extended to TM1 and TM2 of MotA, using Cys residues introduced in several positions in the segments. The observed patterns of disulfide cross-linking indicate that the TM2 segment is positioned between segments TM3 and TM4 of the same subunit, where it could contribute to the proton-channel-forming part of the structure. TM1 is at the interface between TM4 of its own subunit and the TM3 segment of another subunit, where it could stabilize the complex. A structural model based on the cross-linking results shows unobstructed pathways reaching from the periplasm to the Asp 32 residues near the inner ends of the MotB segments. The model indicates a close proximity for certain conserved, functionally important residues. The results are used to develop an explicit model for the proton-induced conformational change in the stator.The bacterial flagellar motor is an ion-fueled machine capable of turning at hundreds of revolutions per second (1-3). In most species, the motor can turn either CW or CCW, and reversals in direction are the basis of regulated movement such as chemotaxis (4,5). Although more than two-dozen proteins are needed for assembly and operation of the flagellum, only five are thought to function closely in rotation. FliG, FliM, and FliN form the "switch complex" on the rotor that determines the direction of rotation (6,7). MotA and MotB are membrane proteins that form the stator, functioning to conduct protons across the membrane and couple proton flow to rotation (8-12) (Figure 1, left). While structural studies of the rotor proteins are fairly advanced (13-16), less is known about the structure of the stator. The stator complexes have subunit composition MotA 4 MotB 2 (17,18). MotA has four TM segments, relatively large domains in the cytoplasm, and only short segments in the periplasm (9,19). MotB has a short segment in the cytoplasm, a single TM segment, and a large periplasmic domain that includes features believed to bind peptidoglycan (10,20,21 In a current model for the rotation mechanism, torque is produced as the stator undergoes conformational changes driven by proton association/dissociation at Asp 32 (23). In support of this model, mutations that neutralized the charge of Asp 32 (e.g., replace...

Flagella stator homologs function as motors for myxobacterial gliding motility by moving in helical trajectories

“…MotA, which drive the rotation of the flagella rotor (21,22). In M. xanthus, complexes of AglR, AglQ and AglS serve as motors in gliding motility.…”

Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

“…S1). The atomic structure of MotA/B was constructed based on the disulfide cross-linking (16)(17)(18) and tryptophan scanning mutations (19,20). The dynamic permeation of hydronium ions, sodium ions, and water molecules was observed using a steered molecular dynamics (SMD) simulation (32)(33)(34), and free energy profiles for ion/water permeation were calculated by umbrella sampling.…”

“…The latter corresponds to the B segment and the additional five residues at both ends. The MotA/B complex structure was constructed based on disulfide cross-linking (16)(17)(18) and Trp scanning mutations (19,20) by a method similar to the NMR structure determination with distance restraints (35); however, a more careful procedure was conducted to avoid overfitting and to examine possible inconsistencies that can be caused by potential ambiguities in the structural information. The side-chain to side-chain restraints were applied to the cross-linked residue pairs with significantly high yields (S-S restraints hereafter; 41 Cys mutations, 83 restraints, Dataset S1) using weak restraint forces, which allow relatively large distance violations (SI Text).…”

“…The TM segments were initially arranged in a similar way to that for the model reported by Kim et al (Fig. 1C) (18) and the A1−A2 and A3−A4 loops were modeled. Double Cys mutants of MotA were yielded as their dimers and tetramers (17,18).…”

“…Systematic Cys and Trp mutagenesis (16)(17)(18)(19)(20) has provided essential information on the structure and function of the flagellar motor. Each MotA (295 residues) contains four transmembrane (TM) alpha helical segments (A1-A4), two short loops in the periplasm, and two long segments (residues 61-160 and 228-295) in the cytoplasm (Fig.…”