The TolQRA proteins of Escherichia coli form an inner membrane complex involved in the maintenance of the outer membrane stability and in the late stages of cell division. The TolQR complex uses the proton-motive force to regulate TolA conformation and its interaction with the outer membrane Pal lipoprotein. It has been proposed that an ion channel forms at the TolQR transmembrane helix interface. This complex assembles with a minimal TolQ/TolR ratio of 4:2, therefore involving at least 14 transmembrane helices, which may form the ion pathway. The C-terminal periplasmic domain of TolR protein interacts with TolQ and has been proposed to control the TolQR channel activity. Here, we constructed unique cysteine substitutions in the last 27 residues of TolR. Each of the substitutions results in a functional TolR protein. Disulfide cross-linking demonstrates that the TolQR complex is dynamic, involving conformational modifications of TolR C-terminal domain. We monitored these structural changes by cysteine accessibility experiments and showed that the conformation of this domain is responsive to the proton-motive force and on the presence of critical residues of the ion pathway.
The TolQRA proteins of Escherichia coli form an inner membrane complex involved in the maintenance of the outer membrane stability and in the late stages of cell division. The TolQR complex uses the proton motive force to regulate TolA conformation and its interaction with the outer membrane Pal lipoprotein. It has been proposed that an ion channel forms at the TolQR transmembrane helix (TMH) interface. This complex assembles with a minimal TolQ:TolR ratio of 4 -6:2 and therefore involves 14 -20 TMHs. To define the organization of the transmembrane helices in the membrane within the TolQR complex, we initiated a cysteine scanning study. In this study, we report results for the systematic replacement of each residue of the TolR TMH. Phenotypic analyses first showed that most of the mutants are functional. Three mutants, TolR L22C, D23C, and V24C, were shown to affect TolQR functioning. Disulfide bond complex formation further showed that two TolR anchors are close enough to interact. Two substitutions, L22C and V24C, form high level of dimers, suggesting that the TolR helix rotates as molecular gears between these two positions and that disulfide bond formation between these residues blocked the rotary motion. Mutations of critical residues located within the TolQ TMH2 and TMH3 and the TolR TMH and proposed to form the ion pathway prevent rotation between these two residues. TolR anchors may form molecular gears that oscillate in response to proton motive force to regulate channel activity.
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