Abstract
The binding protein-dependent maltose transport system of enterobacteria (MalFGK(2)), a member of the ATP-binding cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and of two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. In addition, an extracellular (periplasmic) substrate-binding protein (MalE) is required for activity. Ligand translocation and ATP hydrolysis are dependent on a signaling mechanism originating from the binding protein and traveling through MalF/MalG. Thus, subunit-subunit interactions in the complex are crucial to the transport process but the chemical nature of residues involved is poorly understood. We have investigated the proximity of residues in a conserved sequence ("EAA" loop) of MalF and MalG to residues in a helical segment of the MalK subunits by means of site-directed chemical cross-linking. To this end, single cysteine residues were introduced into each subunit at several positions and the respective malF and malG alleles were individually co-expressed with each of the malK alleles. Membrane vesicles were prepared from those double mutants that contained a functional transporter in vivo and treated with Cu(1,10-phenanthroline)(2)SO(4) or bifunctional cross-linkers. The results suggest that residues Ala-85, Lys-106, Val-114, and Val-117 in the helical segment of MalK, to different extents, participate in constitution of asymmetric interaction sites with the EAA loops of MalF and MalG. Furthermore, both MalK monomers in the complex are in close contact to each other through Ala-85 and Lys-106. These interactions are strongly modulated by MgATP, indicating a structural rearrangement of the subunits during the transport cycle. These data are discussed with respect to current transport models.
Highlights
The binding protein-dependent maltose transport system of enterobacteria (MalFGK2), a member of the ATPbinding cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and of two copies of an ATPase subunit, MalK, which hydrolyze ATP, energizing the translocation process
Of S. typhimurium fully retained its function in maltose transport [16]
The resulting triple transformants are capable of utilizing maltose as sole source of carbon and energy, as indicated by their dark red appearance on McConkey/maltose agar (Fig. 2), clearly suggesting that none of the native cysteine residues of the MalFGK2 complex is essential for function
Summary
Bacterial Strains and Plasmids—E. coli strain ED169 (FϪ ⌬lacU169 araD139 rpsL relA thi flbB ⌬malB107) is described elsewhere [13]. Membrane Preparations—Cells of strain ED169 cotransformed with plasmids encoding mono-Cys variants of MalK (derivatives of pSU19) and mono-Cys variants of MalF and MalG, respectively (derivatives of pTZ18R), were grown in LB medium supplemented with ampicillin (100 g/ml) and chloramphenicol (20 g/ml) at 30 °C to an OD650 ϭ 0.3. Cross-linking Catalyzed by Cu(1,10-phenanthroline)2SO4 (CuPhe)— If not stated otherwise, membrane vesicles at a final concentration of 1 mg/ml in 20 mM Tris-HCl, pH 7.0, containing 5 mM MgCl2 (assay buffer) were routinely treated with CuPhe (3 mM CuSO4/9 mM 1,10-phenanthroline) for 30 min on ice. The reaction was terminated by the addition of 5ϫ SDS sample buffer, containing 5 mM N-ethylmaleimide. For studying the effect of ATP on the cross-linking reaction, membrane vesicles were first incubated in buffer 2, containing 10 mM EDTA for 30 min on ice, collected by ultracentrifugation, washed once in assay buffer containing 5 mM DTT, and resuspended in 20 mM Tris-HCl, pH 7.0. N,NЈo-Phenylenedimaleimide and N,NЈ-p-phenylenedimaleimide were purchased from Sigma
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