Trapping the transition state of an ATP-binding cassette transporter: evidence for a concerted mechanism of maltose transport

Abstract

High-affinity uptake into bacterial cells is mediated by a large class of periplasmic binding protein-dependent transport systems, members of the ATP-binding cassette superfamily. In the maltose transport system of Escherichia coli, the periplasmic maltose-binding protein binds its substrate maltose with high affinity and, in addition, stimulates the ATPase activity of the membrane-associated transporter when maltose is present. Vanadate inhibits maltose transport by trapping ADP in one of the two nucleotide-binding sites of the membrane transporter immediately after ATP hydrolysis, consistent with its ability to mimic the transition state of the gamma-phosphate of ATP during hydrolysis. Here we report that the maltose-binding protein becomes tightly associated with the membrane transporter in the presence of vanadate and simultaneously loses its high affinity for maltose. These results suggest a general model explaining how ATP hydrolysis is coupled to substrate transport in which a binding protein stimulates the ATPase activity of its cognate transporter by stabilizing the transition state.

Figure 1

Analysis of vanadate-treated proteoliposomes. Purified MalFGK₂ (2.5 μM) (18), reconstituted in proteoliposomes, was incubated with 5 μM purified MBP (21), 14 mM MgCl₂, 4 mM ATP, and 0.1 mM maltose for 20 min at 37°C in the presence (●) or absence (○) of 0.5 mM vanadate. Proteoliposomes were then diluted 20-fold in 20 mM Hepes (pH 8)/0.1 mM EDTA, collected by centrifugation, and suspended in Hepes/EDTA buffer. (A) [¹⁴C]Maltose uptake into proteoliposomes (see Materials and Methods). (B) ATPase activity (see Materials and Methods) is measured on the same freeze/thawed proteoliposome preparation that was used for the transport measurements. (Proteoliposomes were washed by centrifugation to remove excess unlabeled ATP before assay.) (C) The protein content of proteoliposome pellets is visualized by Coomassie staining after SDS/PAGE. Lanes 1–3: Proteoliposomes incubated in the presence or absence of vanadate and ATP as indicated. Lane 4: Sample treated as in lane 1, but with 20 μM MBP.

Figure 2

Formation of a stable complex between MBP and MalFGK₂ in detergent solution. MalFGK₂ (2.5 μM) in 20 mM Hepes (pH 8), 10% glycerol, and 0.01% n-dodecyl-β-d-maltoside (exhibiting 0.2 μmol/min/mg of ATPase activity) was incubated with 5 μM MBP, 4 mM MgCl₂, and 4 mM ATP for 20 min at 37°C in the presence (●) or absence (○) of 0.5 mM vanadate, then loaded onto a HiTrap Q Sepharose column (Amersham Pharmacia) and eluted with a gradient of NaCl. (A) OD₂₈₀ of fractions eluting during the gradient. (B) Radioactive elution profile of a sample incubated in the presence of 1 mM MgCl₂ and 0.8 mM [α-³²P]ATP in place of unlabeled ATP. The sample is desalted before ion exchange. (C) Radioactive elution profile of a sample incubated in the presence of 0.5 mM vanadate and 10 μM [¹⁴C]maltose in place of unlabeled maltose. The sample is desalted before ion exchange. (D) Protein composition of fractions from a sample incubated in the absence of vanadate, visualized by Coomassie staining after SDS/PAGE. L, loaded sample. (E) Protein composition of fractions from a sample incubated in the presence of vanadate.

Figure 3

Gel filtration of the MBP-MalFGK₂ complex. After ion-exchange chromatography, fractions containing peak B (Fig. 2E) were pooled, dialyzed against 20 mM Hepes (pH 8.0) and 0.01% n-dodecyl-β-d-maltoside, and stored for 30 days at −70°C. This purified material was thawed and subjected to gel filtration chromatography on a Superose 6 column (Amersham Pharmacia). (A) Elution profile of the gel filtration column. (B) Protein composition of peak fractions from A, visualized by SDS/PAGE and Coomassie staining of 8–25% gradient gels. Lane 1, Molecular weight markers. Lanes 2–6, Contiguous fractions across the emission peak. The expected position of elution of free MBP is indicated by the arrow.

Figure 4

A model for maltose transport. (A) MBP binds maltose and undergoes a conformational change from an open to a closed conformation, generating a high-affinity sugar-binding site. In the closed conformation, MBP binds MalFGK₂ to initiate transport and hydrolysis. (B) In the transition state, MBP and MalFGK₂ become tightly bound to each other, and both proteins have opened, exposing internal binding sites to each other. Opening of MBP in the transition state weakens the interaction between MBP and maltose, facilitating the transfer of sugar to the low-affinity binding site in MalFGK₂. (C) Maltose is transported and MBP is released after the re-exposure of the internal binding site to the cytoplasm. The MalK subunits are modeled after the Rad50 catalytic domain structure (33). This ABC protein undergoes an ATP-induced dimerization and activation step that completes both nucleotide-binding sites with residues donated from the opposing subunit. By analogy, MBP may stimulate the ATPase activity of MalK by bringing the two subunits into close proximity in the transition state.