Mapping the Interactions between Escherichia coli Tol Subunits

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.

Mapping the Interactions between Escherichia coli TolQ Transmembrane Segments

“…The difficulty to draw a clear and comprehensible static model can be inferred from the facts that (i) several of these TMHs might be twisted, kinked, or arranged in a non-parallel organization and (ii) these proteins are probably involved in a highly dynamic process involving periodical TMH rearrangements. Indeed, the TolR anchor (and the corresponding TM segment of MotB) has been previously shown to rotate (19,24), whereas large conformational modifications of MotA have been evidenced by limited proteolysis (21).…”

“…Routinely, cells were grown aerobically in LB medium at 37°C supplemented with ampicillin (100 g/ml) and/or kanamycin (50 g/ml) when required. Plasmid pOK-Q HA and pQ8R encoding functional TolQ proteins containing a C-terminal hemagglutinin (HA) and a C-terminal 8-histidine tag, respectively, and pUC-R encoding TolR, have been described elsewhere (24,38). pOK-T7Q8R was constructed using pOK-QR (24) as template by two sequential PCRs: (i) insertion of the sequence encoding the 8-histidine tag to tolQ (using the same primers as previously used for pQ8R) and (ii) the addition of the sequence of the T7 promoter at the XbaI site upstream of the lac operator sequence using primers: 5Ј-CCTATAGTGAGTCGTATTAAtctagaATACACAACATACGA-GCCGCG-3Ј and 5Ј-ATtctagaTTAATACGACTCACTAT-AGGGGAATTGTGAGCGGATAACAATTTCACACAGG-3Ј (XbaI site in lowercase letters, T7 promoter underlined).…”

“…In Vivo Disulfide Bond Formation and ImmunodetectionCysteine scanning was essentially carried out as described previously (22,24) with slight modifications. 8 ϫ 10…”

“…ExbBD and MotAB convert the chemical energy from the ion or proton gradient of the inner membrane to mechanical movements that lead to import of siderophores and to flagellar rotation, respectively (21,23). Conformational changes in response to PMF have been demonstrated in these different complexes: rotation of the MotB and TolR transmembrane helices (19,24) and structural transition of the MotA cytoplasmic domain and of the TolR, ExbD, TonB, and TolA periplasmic domains (21,22,25,26). In addition to PMF, the structural modifications of TonB and TolA have been shown to be dependent upon the ExbBD and TolQR proteins, respectively (25,26).…”

Directional intracellular trafficking in bacteria

“…H + -flux through ExbB/ ExbD induces conformational changes in TonB that are converted to conformational changes in TonB-dependent receptors in the outer membrane, such as FhuA, to energize transport over this membrane. TolQ and TolR together with TolA form a complex in the cytoplasmic membrane that is important for outer membrane integrity and cell division (10). Here, H + -flux through the TolQ/TolR H + -channel induces conformational changes in TolA, which ultimately changes the interaction between TolA and the outer membrane protein Pal (10).…”