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The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites.

Siarheyeva A, Sharom FJ - Biochem. J. (2009)

Bottom Line: The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered.The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity.These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

ABSTRACT
MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA-MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA-MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35-10 microM. Lipid A and daunorubicin were able to bind to MsbA-MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter.

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Quenching of MsbA–MIANS fluorescence by nucleotidesIncreasing concentrations of nucleotides were added to 100 μg/ml MsbA–MIANS in 0.05% (w/v) DM buffer at 23 °C (with exception of ATP, which was added to MsbA at 10 °C to prevent ATP hydrolysis). The fluorescence emission was monitored at 420 nm (λex=322 nm). The percent quenching of MsbA–MIANS fluorescence (ΔF/F0×100) was calculated relative to the fluorescence in the absence of nucleotides. The continuous line represents the best computer-generated fit of the data points to an equation describing interaction with a single type of binding site, and were used to estimate the Kd of binding. (A) ATP, (B) AMP, (C) ADP and (D) ATP[S] (ATPγS). Data points are the means±S.D. (n=3).
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Figure 8: Quenching of MsbA–MIANS fluorescence by nucleotidesIncreasing concentrations of nucleotides were added to 100 μg/ml MsbA–MIANS in 0.05% (w/v) DM buffer at 23 °C (with exception of ATP, which was added to MsbA at 10 °C to prevent ATP hydrolysis). The fluorescence emission was monitored at 420 nm (λex=322 nm). The percent quenching of MsbA–MIANS fluorescence (ΔF/F0×100) was calculated relative to the fluorescence in the absence of nucleotides. The continuous line represents the best computer-generated fit of the data points to an equation describing interaction with a single type of binding site, and were used to estimate the Kd of binding. (A) ATP, (B) AMP, (C) ADP and (D) ATP[S] (ATPγS). Data points are the means±S.D. (n=3).

Mentions: MIANS fluorescence quenching experiments were carried out on purified MsbA–MIANS in 0.05% (w/v) DM buffer. Saturable concentration-dependent quenching of MsbA–MIANS fluorescence was observed on addition of several drugs, including valinomycin, vinblastine, verapamil, daunorubicin and quercetin (Figure 7), nucleotides (Figure 8), and the putative physiological substrate, lipid A (Figure 9). The maximal percentage of quenching depended on the particular compound, and varied from 6 to 60% (Table 2), with the highest quenching noted for daunorubicin and the lowest for ATP[S] (adenosine 5´-[γ-thio]triphosphate). No significant shift in the λem for MsbA–MIANS was detected in the presence of any of these compounds. To examine MsbA interactions with drugs and nucleotides quantitatively, analysis of the fluorescence quenching titration experiments was carried out. For all drugs and nucleotides tested, the change in MIANS fluorescence followed a hyperbolic curve and was monophasic (Figures 7–9), which suggests the existence of only one binding site, or possibly two binding sites of similar affinity. Curve-fitting was used to estimate the dissociation constant, Kd, and the maximal percentage of quenching, (ΔFmax/Fo×100), for each drug. The estimated Kd values obtained for amphipathic drugs ranged from 0.35 to 8.56 μM (Table 2), and are comparable with the affinity estimated for the physiological substrate, lipid A (5.46 μM). To test the effect of a lipid bilayer on MsbA drug binding affinity, the purified protein was reconstituted into egg PC proteoliposomes by detergent destabilization. The binding affinity of daunorubicin was essentially the same for MsbA in DM buffer and lipid bilayers (Table 2). The Kd for nucleotides varied from 0.13 to 3.5 mM, with the exception of ATP[S], which bound to MsbA with a relatively high affinity of 1.82 μM. ATP binding was measured at 10 °C to avoid hydrolysis of the nucleotide. At this temperature, the Kd of MsbA–MIANS for ATP binding was >7-fold higher than that determined for the non-hydrolysable analogue p[NH]ppA at 23 °C (Table 2).


The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites.

Siarheyeva A, Sharom FJ - Biochem. J. (2009)

Quenching of MsbA–MIANS fluorescence by nucleotidesIncreasing concentrations of nucleotides were added to 100 μg/ml MsbA–MIANS in 0.05% (w/v) DM buffer at 23 °C (with exception of ATP, which was added to MsbA at 10 °C to prevent ATP hydrolysis). The fluorescence emission was monitored at 420 nm (λex=322 nm). The percent quenching of MsbA–MIANS fluorescence (ΔF/F0×100) was calculated relative to the fluorescence in the absence of nucleotides. The continuous line represents the best computer-generated fit of the data points to an equation describing interaction with a single type of binding site, and were used to estimate the Kd of binding. (A) ATP, (B) AMP, (C) ADP and (D) ATP[S] (ATPγS). Data points are the means±S.D. (n=3).
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Figure 8: Quenching of MsbA–MIANS fluorescence by nucleotidesIncreasing concentrations of nucleotides were added to 100 μg/ml MsbA–MIANS in 0.05% (w/v) DM buffer at 23 °C (with exception of ATP, which was added to MsbA at 10 °C to prevent ATP hydrolysis). The fluorescence emission was monitored at 420 nm (λex=322 nm). The percent quenching of MsbA–MIANS fluorescence (ΔF/F0×100) was calculated relative to the fluorescence in the absence of nucleotides. The continuous line represents the best computer-generated fit of the data points to an equation describing interaction with a single type of binding site, and were used to estimate the Kd of binding. (A) ATP, (B) AMP, (C) ADP and (D) ATP[S] (ATPγS). Data points are the means±S.D. (n=3).
Mentions: MIANS fluorescence quenching experiments were carried out on purified MsbA–MIANS in 0.05% (w/v) DM buffer. Saturable concentration-dependent quenching of MsbA–MIANS fluorescence was observed on addition of several drugs, including valinomycin, vinblastine, verapamil, daunorubicin and quercetin (Figure 7), nucleotides (Figure 8), and the putative physiological substrate, lipid A (Figure 9). The maximal percentage of quenching depended on the particular compound, and varied from 6 to 60% (Table 2), with the highest quenching noted for daunorubicin and the lowest for ATP[S] (adenosine 5´-[γ-thio]triphosphate). No significant shift in the λem for MsbA–MIANS was detected in the presence of any of these compounds. To examine MsbA interactions with drugs and nucleotides quantitatively, analysis of the fluorescence quenching titration experiments was carried out. For all drugs and nucleotides tested, the change in MIANS fluorescence followed a hyperbolic curve and was monophasic (Figures 7–9), which suggests the existence of only one binding site, or possibly two binding sites of similar affinity. Curve-fitting was used to estimate the dissociation constant, Kd, and the maximal percentage of quenching, (ΔFmax/Fo×100), for each drug. The estimated Kd values obtained for amphipathic drugs ranged from 0.35 to 8.56 μM (Table 2), and are comparable with the affinity estimated for the physiological substrate, lipid A (5.46 μM). To test the effect of a lipid bilayer on MsbA drug binding affinity, the purified protein was reconstituted into egg PC proteoliposomes by detergent destabilization. The binding affinity of daunorubicin was essentially the same for MsbA in DM buffer and lipid bilayers (Table 2). The Kd for nucleotides varied from 0.13 to 3.5 mM, with the exception of ATP[S], which bound to MsbA with a relatively high affinity of 1.82 μM. ATP binding was measured at 10 °C to avoid hydrolysis of the nucleotide. At this temperature, the Kd of MsbA–MIANS for ATP binding was >7-fold higher than that determined for the non-hydrolysable analogue p[NH]ppA at 23 °C (Table 2).

Bottom Line: The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered.The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity.These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

ABSTRACT
MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA-MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA-MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35-10 microM. Lipid A and daunorubicin were able to bind to MsbA-MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter.

Show MeSH
Related in: MedlinePlus