Abstract
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.
Highlights
ROTATION OF THE TolR TRANSMEMBRANE HELIX *It has been proposed that an ion channel forms at the TolQR transmembrane helix (TMH) interface
The Tol proteins form a complex involved in the maintenance of cell envelope integrity [1, 2] and in the late stage of the cell division process [3]
To understand how the TolQRA TM segments interact in the membrane and how they mechanically respond to proton motive force (PMF) or ion transit, we initiated a cysteine scanning approach
Summary
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 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. The Tol proteins form a complex involved in the maintenance of cell envelope integrity [1, 2] and in the late stage of the cell division process [3] Three of these proteins, TolQ, TolR, and TolA interact through their transmembrane (TM) segments in the inner membrane (4 – 8). The proline residue at position 187 of TolQ (TMH3) is essential and has been proposed to regulate conformational modifications of the TolQR complex in response to ion transit through the aqueous channel [14]. Further experiments support the model in which ion transit at the TolQR TM helices interface induce a rotary motion of the TolR TM segment between the two faces
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