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. 2013 Jul 26;288(30):21638-47.
doi: 10.1074/jbc.M113.485714. Epub 2013 Jun 13.

Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA

Rupak Doshi  1 , Hendrik W van Veen
Affiliations

Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA

Rupak Doshi et al. J Biol Chem. .

Abstract

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.

Keywords: ABC Transporter; Lipid Transport; Membrane Transport; Multidrug Transporters; Protein Conformation; Substrate-stimulated ATPase.

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Figures

FIGURE 1.
FIGURE 1.
Substrate binding by the MsbA dimer causes NBD dimerization. a and b, cysteine-less MsbA containing a single cysteine residue near the NBD (E208C MsbA-cl) shows a substrate-dependent enhancement in the intensity of E208C-E208C′ cross-linked MsbA dimers (D) in ISOVs. To study the conformational changes due to substrate binding, ISOVs containing E208C MsbA-cl were subjected to cysteine cross-linking in the presence of the substrates verapamil or Hoechst 33342 (both in water) or lipid A (controlled against an equal volume of the solvent DMSO). Briefly, ISOVs at 5–7 mg/ml total membrane protein, in 100 mm K-HEPES buffer, pH 7.0, containing 5 mm MgSO4 were first mixed with 0.5 mm DTT for 2 min at 20 °C to reduce preformed cross-links. Substrate was then added at the indicated concentrations. Following 5 min of incubation at 20 °C, cross-linking was initiated by the addition of 0.5 mm CuPhen. After 5 min of incubation in a 30 °C shaker incubator, reactions were stopped by the addition of 10 mm NEM. 10–15 μg of protein from each sample was mixed with 6× SDS sample loading buffer devoid of reducing agents, separated on a 10% SDS-polyacrylamide gel, and transferred to Western blot probed with anti-His tag antibody. This assay was performed previously in the presence of nucleotides instead of substrates (13, 14). c, densitometry on dimer bands is presented as -fold change in intensity relative to the no substrate control for verapamil or Hoechst 33342 and relative to the DMSO control for lipid A (**, p < 0.03; *, p < 0.05 Student t test; n = 3, error bars represent mean ± S.E.).
FIGURE 2.
FIGURE 2.
Analyses of FRET between Atto-dye-labeled E208C and E208C′ in the MsbA-cl dimer. a and b, Ni2+ affinity resin-bound E208C MsbA-cl was labeled with Atto590 (donor, D) or Atto665 (acceptor, A) or both (DA). To test whether the labeling reaction was complete (and that no free labels/unreacted thiol groups were present in eluted samples), 1 μg of the purified labeled protein fractions, with or without excess (5 mm) CuPhen, was analyzed by SDS-PAGE in the absence of DTT. Coomassie Brilliant Blue staining (A) shows total protein in the sample (Mo, monomers; Di, dimers) and followed analysis of the gel by in-gel UV fluorescence (b) to detect D-labeled monomers. a and b represent the same gel and are cropped to enhance readability. c, band intensities in b were measured using densitometry and are presented as percentage of monomers labeled with D. Labeling of E208C-D was set at 100%. d, at the excitation maximum for D (594 nm), buffer (trace 1) or DA in the absence of protein (trace 2) did not produce any noticeable fluorescence at the emission maximum for A (680 nm). e, such a signal was also not obtained for E208C-D (trace 1) and E208C-A (trace 2), whereas clear evidence for transfer of fluorescence energy from D to A under these conditions was observed for E208C-DA (trace 4). In another control experiment, fluorescence emission by A was also observed for E208C-A at the excitation maximum of A (663 nm) (trace 3). f, MsbA-cl mixed with DA did not yield any FRET signal at 680 nm (trace 1). The addition of lipid A (40 μg/ml) did not affect fluorescence at 680 nm (trace 2). Similarly, the addition to E208C-A of the lipid A or ATP plus sodium orthovanadate (2 mm each, to trap MsbA with ADP·Vi) or 2 mm ADP did not cause any appreciable alterations in the fluorescence emission at 680 nm (traces 3–6).
FIGURE 3.
FIGURE 3.
FRET between E208C-DA demonstrates NBD closure upon substrate binding. a, addition of lipid A (40 μg/ml) or 2 mm ADP·Vi to E208C-DA led to significant elevations in the FRET intensity at 680 nm, compared with equal volume of the assay buffer or DMSO (solvent) or 2 mm ADP. b, each ligand condition was replicated three times with protein, purified, and labeled from independently prepared batches of ISOVs. Relative intensities at 680 nm compared with the buffer condition are shown (**, p < 0.04; n = 3, mean ± S.E.).
FIGURE 4.
FIGURE 4.
Substrate binding by MsbA does not cause MD separation. a and b, cysteine cross-linking-Atto590 labeling with A281C, which is located at the extracellular side of the MDs. We previously used this reaction to detect nucleotide-dependent conformational changes in the MsbA dimer in ISOVs (14). Here, we did not observe any marked changes in the proportion of dimers (D, dimers visualized on Coomassie Blue-stained SDS-polyacrylamide gels) or Atto590-labeled monomers (M, monomers visualized through in-gel UV fluorescence from Atto590) when increasing concentrations of the substrates verapamil, Hoechst 33342, or lipid A, or the solvent controls were included in the reaction instead of nucleotides. In contrast, incubations with AMP-PNP served as a robust positive control for the detection of MD separation in the outward-facing state, and gave clear alterations in dimer and monomer band intensities. c, densitometry on dimer (left) and monomer (right) signals is presented as -fold change in intensity relative to the no substrate/nucleotide control, which was taken as 1 in each experiment (**, p < 0.04 AMP-PNP versus verapamil/Hoechst 33342/lipid A; n = 3, mean ± S.E.).
FIGURE 5.
FIGURE 5.
Confirmation of the disparity between substrate-dependent cross-linking between E208C-E208C′ and A281C-A281C′ in MsbA-cl. a and b, the lowest verapamil concentration (5 μm) already enhanced cysteine cross-linked E208C–E208C′ dimers (D) and decreased in Atto590-labeled monomers (M) (n = 3, mean ± S.E.). This assay was performed similar to that detailed under “Materials and Methods,” with the exception that 0.5 mm DTT was first added to reduce pre-formed E208C cross-links, and 0.5 mm CuPhen was added (5 min, 30 °C shaker incubator) prior to the addition of Atto590. c and d, level of cross-linked A281C-A281C′ dimers did not change in the presence of verapamil up to concentrations of 15 μm. No changes in monomer intensities or cross-linked dimer intensities were observed when cysteine cross-linking was performed in the presence of Atto590 (see Fig. 4).
FIGURE 6.
FIGURE 6.
Expression and activity of the double-cysteine mutant E208C/A281C MsbA-cl. a, total membrane proteins in ISOVs harboring the E208C/A281C mutant were separated on an SDS-polyacrylamide gel and analyzed by Western blot. The double mutant protein expressed as well as MsbA-cl and underwent spontaneous Cys-Cys cross-linking in the absence of DTT, similar to the single mutant proteins E208C and A281C (13, 14). The high molecular weight signals arising due to this spontaneous cross-linking could be abolished by the use of DTT in the sample loading buffer. b, both MsbA-cl and the noncross-linked E208C/A281C mutant catalyzed metabolic energy-dependent ethidium export from L. lactis cells that were pre-loaded with 2 μm ethidium bromide. Fluorescence traces are typical for data obtained in three independent experiments using different batches of cells.
FIGURE 7.
FIGURE 7.
Effect of substrate binding on the kinetics of ATP binding and hydrolysis. a, binding curves (with R2 values >0.99) and binding parameters (Kd and Bmax, see text for details) of purified WT MsbA for the fluorescent analog of ATP, TNP-ATP, were unaffected by the inclusion of increasing amounts of verapamil up to 200 μm (n = 3, mean ± S.E.). b, lipid A (100 μg/ml)-stimulated ATPase activity of MsbA-cl was measured over a range of ATP concentrations, after which the data were fitted to a hyperbola (R2 values for both curves were >0.99). An equal volume of DMSO was used as the solvent control in the absence of lipid A. The stimulation of ATPase activity was due to a significant (p < 0.03) ∼1.24-fold increase in the maximum rate of hydrolysis (Vmax), with no observable changes in the apparent affinity for ATP (Km) (see text for parameter details; mean ± S.E., n = 3; error bars, where not visible, are hidden within the symbols).
FIGURE 8.
FIGURE 8.
Proposed conformational changes of the MsbA dimer in the transition from inward-facing to outward-facing. Current mechanistic models for substrate transport by the MsbA dimer suggest substrate binding to the inward-facing conformation of dimeric MsbA (Protein Data Bank code 3B5W, full model) and its transition to the ATP-bound, outward-facing structure (Protein Data Bank code 3B60) (7, 21). The two A281C residues (in orange) are in close proximity in the inward-facing open state, whereas the two E208C residues (in green) are close in the outward-facing state. Our analyses suggest that substrate binding to MsbA in step 1 stabilizes an intermediate state that precedes the outward-facing conformation. In this intermediate state, both pairs of A281C/A281C′ and E208C/E208C′ residues are in close proximity. ATP binding to this intermediate state in step 2 switches MsbA into the outward-facing conformation by allowing the formation of stabilizing tetrahelix bundle interactions (helices in blue and red) (14). ATP hydrolysis is then required to resolve the outward-facing conformation back to an inward-facing conformation. The substrate-bound intermediate with dimerized NBDs as in the outward-facing state, but without separation of the MDs at the external side, might share similarities with the crystal structure of inward-facing closed MsbA (Protein Data Bank code 3B5X) (7), although the resolution of this structure is too low to allow accurate predictions of Cys-Cys cross-linking.
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