Arrangement of Core Membrane Segments in the MotA/MotB Proton-Channel Complex of Escherichia coli

Abstract

The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.

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).…”

“…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). The MotA/B complex contains four MotA proteins; therefore, all of the possible combinations of the TM segment pairs were considered for the dimer-forming S-S restraints, which resulted in eight distinct initial models (groups 1-8; see Fig.…”

“…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.…”

“…The B segment is expected to form a proton channel together with A3 and A4 (Fig. 1C) (17). Only a few polar residues have been identified in the predicted TM segments of MotA/B (19,20), which implies that the channel surface should be relatively hydrophobic.…”