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. 2013 Mar 8;288(10):6801-13.
doi: 10.1074/jbc.M112.430074. Epub 2013 Jan 10.

Molecular disruption of the power stroke in the ATP-binding cassette transport protein MsbA

Rupak Doshi  1 , Anam Ali, Wilma Shi, Elizabeth V Freeman, Lisa A Fagg, Hendrik W van Veen
Affiliations

Molecular disruption of the power stroke in the ATP-binding cassette transport protein MsbA

Rupak Doshi et al. J Biol Chem. .

Abstract

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.

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Figures

FIGURE 1.
FIGURE 1.
MsbA structures and the tetrahelix bundle. A, nucleotide-free inward-facing structure of dimeric MsbA (Protein Data Bank code 3B5W, full model). B, AMP-PNP-bound outward-facing structure (Protein Data Bank code 3B60) (12). Residues Thr-121–Glu-133 in TM 3 and TM 3′ are in blue, and Asn-195–His-214 in TM 4 and TM 4′ are in red. C, zoomed in snapshot of the tetrahelix bundle viewed from the cytoplasmic side, after rotation of the MsbA dimer in B by 90°. Residues Ser-324 to Gln-582 in each monomer (containing the NBD) are not shown for clarity of presentation. Residues Glu-208 and Lys-212 (in the cytoplasmic extensions of TM 4 and TM 4′) are shown as purple and yellow sticks, respectively. D, residues Ala-281–Ala-281′ (red spheres) in dimeric MsbA are in close proximity in the nucleotide-free inward-facing conformation and distant in the AMP-PNP-bound outward-facing state, whereas Glu-208 to Glu-208′ residues (purple spheres) follow the opposite pattern. For further explanation, see under “A281C-A281C′ Cross-linking in Dimeric MsbA-cl Responds to Nucleotides.”
FIGURE 2.
FIGURE 2.
Expression and purification of MsbA mutants. A, Western blot analysis shows that E208A MsbA, K212A MsbA, and E208A/K212A MsbA mutants were equally well expressed as WT MsbA in the plasma membrane. B, Coomassie-stained SDS-polyacrylamide gel showing the apparent homogeneity of our preparations of purified protein. Approximate positions of the molecular mass marker are shown alongside in A and B.
FIGURE 3.
FIGURE 3.
Effects of alanine mutations in the tetrahelix bundle on active substrate transport and substrate binding. A, when 0.25 μm Hoechst 33342 was added to WT MsbA, E208A MsbA, K212A MsbA, or E208A/K212A MsbA-containing ISOVs, followed by 2 mm ATP, all mutants displayed strongly reduced abilities to transport Hoechst 33342 compared with WT (n = 3). The double mutant E208A/K212A was more severely affected than the single mutants E208A and K212A. B, E208A MsbA, K212A MsbA, and E208A/K212A MsbA were also defective in mediating active export of ethidium from intact cells. Energy-depleted cells were pre-loaded with 2 μm ethidium, and at the arrow, 28 mm glucose was added to provide metabolic energy. Again, the double mutant E208A/K212A showed a larger defect than the single mutants E208A and K212A. Traces in A and B represent observations in at least three independent experiments. C, purified mutant proteins had WT-like Hoechst 33342 binding affinities (n = 3, see text).
FIGURE 4.
FIGURE 4.
Alanine mutations in the tetrahelix bundle do not denature MsbA. WT MsbA and the mutant proteins showed similar overall protein folding that is different from heat-denatured MsbA, as deduced from intrinsic tryptophan fluorescence and trypsin digestion patterns. A, normalized spectral profiles of intrinsic tryptophan fluorescence measured for MsbA WT, without (black trace, labeled WT MsbA) or with (gray trace) denaturation for 1 min at 50 °C, revealed a denaturation-dependent right-shift in the emission maximum. B and C, alanine mutants E208A MsbA and K212A MsbA and the E208A/K212A double mutant gave overlapping tryptophan fluorescence traces (B) with similar peak fluorescence intensities at 328 nm as WT MsbA (n = 4) (C), and it did not show any signs of protein denaturation. Data are elution buffer-subtracted. Error bars represent mean ± S.E. D, trypsin digestions of purified MsbA proteins were examined on 12% SDS-PAGE (9 μg of protein/lane). Lane 1, molecular mass marker; lane 2, WT MsbA; lane 3, WT MsbA + trypsin; lane 4, E208A MsbA + trypsin; lane 5, K212A MsbA+ trypsin; lane 6, E208A/K212A MsbA + trypsin; lane 7, WT MsbA (denatured for 1 min at 50 °C) + trypsin. White triangles refer to major signals for WT MsbA (31 and 7.5 kDa) that were absent for heat-denatured MsbA. Band patterns are typical for observations in four independent experiments.
FIGURE 5.
FIGURE 5.
MsbA mutants used in this study exhibit WT-like binding affinities for nucleotides. TNP-ATP binding and ADP-dependent and ATP-dependent TNP-ATP displacement (inset) were determined in triplicate (n = 3) using purified MsbA proteins in detergent solution (large symbols for TNP-ATP binding and its displacement by ADP; small open and closed circles for displacement of TNP-ATP binding by ATP). ATP concentration scale is ×50 the actual ATP concentration. The IC50 values obtained in the TNP-ATP displacement experiments were used in Equation 3 under “Binding Assays” to calculate the dissociation constants for binding of ADP (KdADP) and ATP (KdATP). Values for KdADP, KdATP, and the dissociation constant for binding of TNP-ATP (KdTNP-ATP) are listed in the text and in Table 1. Error bars represent mean ± S.E.
FIGURE 6.
FIGURE 6.
E208Q mutation partially restores reduced catalytic activity of the E208A mutant. A, although the basal ATP hydrolysis activity of E208A MsbA at 1 mm ATP was reduced to 16.2 ± 3.9% of WT MsbA, E208Q MsbA was able to improve this performance to 44.4 ± 10.0% (100% activity of WT in these experiments equaled 158.5 ± 37.3 nmol ATP/min/mg) (n = 3; *, p < 0.05, Student's t test E208A mutant versus E208Q mutant). B, measurement of the basal ATP hydrolysis activity (n = 3) of detergent-purified WT MsbA (●) and E208A MsbA (○) over a range of ATP concentrations between 0.1 and 4 mm, and display of the data in a Lineweaver-Burk plot, revealed changes in the maximum rate of hydrolysis (Vmax) and Michaelis constant (Km) for E208A MsbA compared with WT MsbA, whereas the Vmax/Km ratio (= 1/slope) was relatively similar. See text for further details. C, inclusion of the MsbA substrate Taxol (32) stimulated the ATPase activity for WT MsbA but not for E208A MsbA. Experimental conditions and 100% value are as given in A, and symbols are as given in B. D, under conditions identical to those described for Fig. 3B, the E208Q mutant mediated the export of ethidium from intact cells. E, initial rates of ethidium efflux were measured over the first 50 s after the addition of glucose. These analyses showed that although E208Q MsbA could not efflux as well as the WT protein, its ethidium extrusion rate was significantly higher than that for E208A MsbA (WT versus E208A MsbA, *, p < 0.05; E208A MsbA versus E208Q MsbA, p < 0.09, Student's t tests). F, E208Q mutation improved Hoechst 33342 transport by MsbA in ISOVs compared with the E208A replacement. Experimental details are similar to those described in the legend to Fig. 3A. Consistent with ethidium transport measurements and ATPase assays, E208Q MsbA was never as efficient as WT MsbA. Traces in D and F and rates in E are based on observations in at least three independent experiments. Error bars represent mean ± S.E.
FIGURE 7.
FIGURE 7.
Cross-linking Atto590 labeling on E208C and A281C in dimeric MsbA-cl. A, when ISOVs containing A281C MsbA-cl were loaded on an SDS-polyacrylamide gel without the reducing agent DTT in the SDS sample-loading buffer and analyzed by Western blot, high molecular weight dimers were observed due to spontaneous cysteine cross-linking. These dimers were absent for MsbA-cl (Cys-less). Cysteine cross-linking was enhanced by incubation with the oxidizing agent copper phenanthroline (CuPhen), although cross-linking disappeared on the inclusion of DTT in the SDS sample-loading buffer. B, under no nucleotide conditions, E208C MsbA-cl (10) or A281C MsbA-cl in ISOVs was subjected to inter-molecular cysteine cross-linking, and MsbA-specific bands (monomers (M)/dimers (D)) were detected on Western blot probed with an anti-penta-His antibody (10). The approximate molecular mass marker positions are shown alongside. C, high saturating levels of Cys-Cys cross-linking were observed for A281C MsbA-cl in the no nucleotide condition. As the reduction in this high intensity after the transition from inward-facing to outward-facing MsbA is associated with a poor signal-to-noise ratio, we combined the reduction in A281C/A281C′ cross-linking with measurements on the increased accessibility of free A281C to the thiol-reactive fluorescent probe Atto590. To validate this method, we re-assayed A281C MsbA-cl and E208C MsbA-cl in the no nucleotide condition. On Coomassie Brilliant Blue staining of the gel, high molecular weight bands corresponding to E208C/E208C′ and A281C/A281C′ Cys-Cys cross-linked dimers (D) were observed, whereas such dimers were not obtained for MsbA-cl. A small but reproducible difference in migration was observed between dimers for the two mutants, which is attributed to anomalous SDS binding. The intensity of dimers for A281C MsbA-cl was found to be higher than for E208C MsbA-cl, consistent with the notion that the MsbA dimer will be in the inward-facing state in the absence of added nucleotides. D, when the same gel was visualized under UV light (prior to Coomassie-staining) only ISOVs containing E208C MsbA-cl or A281C MsbA-cl had the monomers (M) labeled with Atto590; labeled monomers were not observed for control and MsbA-cl ISOVs. Consistent with the enhanced dimer intensity for A281C MsbA-cl, the monomer Atto590-labeling intensity was reduced for this mutant compared with E208C MsbA-cl. The images in C and D pertain to one and the same gel/experiment, repeated at least three times, and were processed in parallel and cropped to increase clarity of presentation.
FIGURE 8.
FIGURE 8.
Nucleotide-responsive cross-linking of A281C MsbA-cl and E208A/A281C MsbA-cl. A, intermolecular A281C-A281C′ cross-linking for A281C MsbA-cl was found to be nucleotide-responsive and thus could be used to monitor conformational changes associated with the ATPase cycle as a complement to previously published work with E208C MsbA-cl (10). All images here pertain to one and the same gel/experiment, processed in parallel to enhance readability (M, monomer; D, dimer). B, Atto590-labeled monomer and Coomassie Brilliant Blue-stained dimer intensities were analyzed by densitometry and are presented as -fold change in intensity relative to the no nucleotide condition (for which the -fold change was set at 1) (n = 3; **, p < 0.03, Student's t test). C, E208A mutation in the A281C background (E208A/A281C MsbA-cl) led to a reduction in Hoechst 33342 transport activity compared with the activity of MsbA-cl and A281C MsbA. A similar reduction was obtained for the E208A mutation in the WT MsbA background (see Fig. 3, A and B). D, compared with A281C MsbA-cl, the E208A/A281C MsbA-cl double mutant displayed a lack of changes in cross-linking Atto590 labeling in response to incubation with the nonhydrolysable ATP analog AMP-PNP (for 6 or 12 min). All panels pertain to one and the same gel/experiment, processed in parallel, and are cropped to enhance readability (M, monomer; D, dimer). E, E208A mutation interrupts the inward- to outward-facing conformational switch in MsbA. Densitometric analysis on the monomer and dimer signals for samples after 12 min of incubation without or with AMP-PNP was as indicated. Data are presented as -fold change in intensity, relative to the no nucleotide condition (for which the -fold change equals 1) (n = 3; *, p < 0.06; **, p < 0.04, Student's t test). Open bars refer A281C MsbA-cl, and gray bars refer to E208A/A281C MsbA-cl. Error bars represent mean ± S.E.
FIGURE 9.
FIGURE 9.
ADP-responsive cross-linking Atto590 labeling of E208C MsbA-cl. A, to study the conformational changes affected by ADP binding, E208C MsbA-cl harboring ISOVs were assayed using cysteine cross-linking Atto590 labeling in the presence of increasing concentrations of ADP. 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 3 min at room temperature to reduce preformed cross-links. ADP was added at the indicated concentrations. Following 3 min of incubation at RT, cross-linking was initiated by the addition of 0.5 mm copper phenanthroline. After 5 min of incubation in a 30 °C shaker incubator, 20 μm Atto590 was added, and the samples were further incubated for 10 min in the 30 °C shaker incubator. Reactions were stopped by the addition of 10 mm N-ethylmaleimide. 10–15 μg of protein from each sample was mixed with 6× SDS sample loading buffer devoid of reducing agents and separated on a 10% SDS-polyacrylamide gel. The gel was first viewed under UV light to detect Atto-labeled monomers (M; bottom panel), followed by Coomassie Brilliant Blue staining to detect dimers (D; top panel). B, monomer/dimer intensities are presented as percentage of total intensity of MsbA-related bands (monomers + dimers), relative to the percentage monomer/dimer intensity observed under the no nucleotide condition (which was set at as 1) (n = 3, data are mean ± S.E.). The results suggest that ADP binding to MsbA induces a conformational shift toward the inward-facing state.
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