A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate

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.

Keywords: ABC transporter; ATPase; X-ray crystallography; bacteria; crystal structure.

Figure 1.

Electrophoretic profiles of oligosaccharides and proteins. A, FACE profiles of alginate oligosaccharides. Lane 1, Δ2M; lane 2, Δ3M; lane 3, Δ4M; lane 4, Δ5M; lane 5, Δ6M; lane 6, Δ7-8M; lane 7, 7–10M; lane 8, 6–8M; lane 9, saturated M oligosaccharides before purification; lane 10, negative control treated with 8-amino-1,3,6-naphthalenetrisulfonic acid in the absence of oligosaccharide. B, SDS-PAGE profiles of AlgM1M2SS (lane 1) and AlgQ2 (lane 2). Three protein bands indicated by arrows correspond to AlgS, AlgM1, and AlgM2 in the purified AlgM1M2SS (lane 1). An arrow indicates the position of AlgQ2 (lane 2). M, mass.

Figure 2.

Co-immunoprecipitation of AlgM1M2SS and AlgQ2. Lane 1, no ligand; lane 2, 2 mm Mg-ATP and 0.2 mm Δ3M; lane 3, 2 mm Mg-ATP, 0.2 mm Δ3M, and 5 mm vanadate. MM, molecular mass.

Figure 3.

In vitro assay of AlgM1M2SS. A, ATPase activity; B, alginate transport activity; activities are shown as relative values. Activities in the absence of oligosaccharides (A) and with Δ4M (B) were taken as 100%. Assays were conducted three times, and data are presented as means with error bars representing S.E.

Figure 4.

Overall structure. The images on the right show the F_o − F_c map contoured at 2.8σ around the alginate oligosaccharide. A, AlgM1M2SS/AlgQ2 + Δ3M; B, AlgQ2 + Δ3M; C, AlgM1M2SS/AlgQ2 + 6–8M; D, AlgQ2 + 7–10M.

Figure 5.

Cross-eyed stereo views of the alginate-binding site of AlgQ2. A, AlgM1M2SS/AlgQ2 + Δ3M; B, AlgQ2 + Δ3M; C, AlgM1M2SS/AlgQ2 + 6–8M; D, AlgQ2 + 7–10M. ΔM and M are shown as yellow and brown saccharides, respectively. Water molecules are shown as blue spheres. Dotted lines, hydrogen bonds.

Figure 6.

A model for ATP hydrolysis by periplasmic solute-binding protein–dependent ABC transporter. The model shows the following steps: (i) resting state before docking of AlgM1M2SS and AlgQ2; (ii) pretranslocation state at the moment of interaction between AlgM1M2SS in the inward-facing conformation and ligand-bound AlgQ2 in the closed conformation; (iii) docking-dependent ATP binding and hydrolysis; and (iv) substrate import or return to resting state in the case of non-transport substrate.