Abstract
The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.
Highlights
ATP-binding cassette (ABC)3 transporters are present in cytoplasmic membranes of all species, and the translocation of substrates by transporters is coupled with ATP hydrolysis [1]
ABC transporters are classified as exporters and importers [3], and whereas ABC exporters are ubiquitous from bacteria to humans, ABC importers are mainly found in prokaryotes [4] and require accessory solute-binding proteins [5]
ABC importers are classified as type I or II based on their structures
Summary
The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solutebinding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. Interactions between the ABC transporter MalFGK2 and the periplasmic maltose-binding protein MalE induce ATP hydrolysis [8]. A mutant MalE was shown to bind sucrose (non-transport ligand) and enhance the ATPase activity of MalFGK2, suggesting that ATP hydrolysis is induced by ligandbound MalE but not by the translocated substrate (i.e. maltose). Two models of solute-binding protein MalE conformations in contact with MalFGK2 were described as closed [6] and open states [9] These findings were based on in vitro assays, and the structure of the ABC transporter in complex with the solute-binding protein with a non-transport ligand has not been directly characterized
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