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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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Catalytic activity of MsbA and MsbA–MIANS(A) The ATPase activity at 37 °C as a function of ATP concentration for MsbA–MIANS (○) and unlabelled MsbA (●). Data points are the means±S.D. (n=3). In both cases, purified MsbA was in a buffer containing 0.05% (w/v) DM. (B) Hill plot of the ATPase activity of wild-type MsbA (●) and MsbA–MIANS (○) as a function of ATP concentration; MsbA Hill coefficient, n=1.03±0.03, MsbA–MIANS Hill coefficient, n=1.01±0.03.
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Figure 2: Catalytic activity of MsbA and MsbA–MIANS(A) The ATPase activity at 37 °C as a function of ATP concentration for MsbA–MIANS (○) and unlabelled MsbA (●). Data points are the means±S.D. (n=3). In both cases, purified MsbA was in a buffer containing 0.05% (w/v) DM. (B) Hill plot of the ATPase activity of wild-type MsbA (●) and MsbA–MIANS (○) as a function of ATP concentration; MsbA Hill coefficient, n=1.03±0.03, MsbA–MIANS Hill coefficient, n=1.01±0.03.

Mentions: Membrane vesicles were isolated from E. coli cells overexpressing His6-tagged wild-type MsbA and the two site-directed mutants, C88S and C315S. After membrane solubilization in DM, MsbA purification was carried out using Ni-NTA chromatography. The fractions containing purified protein were pooled and concentrated, to yield approx. 1 mg of MsbA from 15 mg of membrane protein. Analysis using SDS/PAGE and Western blotting showed that MsbA was isolated at high purity (Figure 1B), with an ATPase-specific activity of 400±5 nmol/min per mg of protein (at 2 mM ATP) for wild-type protein. The ATPase activity of the C88S and C315S mutants did not differ substantially from that of wild-type MsbA. The ATPase activity of wild-type MsbA was measured for a range of ATP concentrations from 0.05–10 mM at 4, 23 and 37 °C (results for 37 °C are shown in Figure 2). Activity increased in a hyperbolic fashion with increasing ATP concentration, and levelled off above 1.5 mM ATP (Figure 2A). Similar ATPase activity dependence on ATP concentration was reported previously [4,7]. The Km for unlabelled MsbA was 0.52 ±0.04 mM, whereas for MIANS-labelled protein (see below) it was 0.41±0.04 mM. The corresponding Vmax values were 418±12 and 354±13 nmol/min per mg of protein, for unlabelled and MIANS-labelled MsbA respectively. We previously reported that purified MsbA is homodimeric in DM solution [14]. When the kinetic data for wild-type and MIANS-labelled MsbA were fitted to the Hill equation, coefficients close to 1 were obtained (Figure 2B), suggesting that the two monomers function independently, and do not interact co-operatively with each other. This is consistent with the “alternating sites” proposal of Senior et al. [34] for the mechanism of Pgp, which is also proposed to be a dual drug transporter and lipid flippase; only one NBD is thought to be active at any instant in time, and the two sites alternate in catalysis.


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

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

Catalytic activity of MsbA and MsbA–MIANS(A) The ATPase activity at 37 °C as a function of ATP concentration for MsbA–MIANS (○) and unlabelled MsbA (●). Data points are the means±S.D. (n=3). In both cases, purified MsbA was in a buffer containing 0.05% (w/v) DM. (B) Hill plot of the ATPase activity of wild-type MsbA (●) and MsbA–MIANS (○) as a function of ATP concentration; MsbA Hill coefficient, n=1.03±0.03, MsbA–MIANS Hill coefficient, n=1.01±0.03.
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Figure 2: Catalytic activity of MsbA and MsbA–MIANS(A) The ATPase activity at 37 °C as a function of ATP concentration for MsbA–MIANS (○) and unlabelled MsbA (●). Data points are the means±S.D. (n=3). In both cases, purified MsbA was in a buffer containing 0.05% (w/v) DM. (B) Hill plot of the ATPase activity of wild-type MsbA (●) and MsbA–MIANS (○) as a function of ATP concentration; MsbA Hill coefficient, n=1.03±0.03, MsbA–MIANS Hill coefficient, n=1.01±0.03.
Mentions: Membrane vesicles were isolated from E. coli cells overexpressing His6-tagged wild-type MsbA and the two site-directed mutants, C88S and C315S. After membrane solubilization in DM, MsbA purification was carried out using Ni-NTA chromatography. The fractions containing purified protein were pooled and concentrated, to yield approx. 1 mg of MsbA from 15 mg of membrane protein. Analysis using SDS/PAGE and Western blotting showed that MsbA was isolated at high purity (Figure 1B), with an ATPase-specific activity of 400±5 nmol/min per mg of protein (at 2 mM ATP) for wild-type protein. The ATPase activity of the C88S and C315S mutants did not differ substantially from that of wild-type MsbA. The ATPase activity of wild-type MsbA was measured for a range of ATP concentrations from 0.05–10 mM at 4, 23 and 37 °C (results for 37 °C are shown in Figure 2). Activity increased in a hyperbolic fashion with increasing ATP concentration, and levelled off above 1.5 mM ATP (Figure 2A). Similar ATPase activity dependence on ATP concentration was reported previously [4,7]. The Km for unlabelled MsbA was 0.52 ±0.04 mM, whereas for MIANS-labelled protein (see below) it was 0.41±0.04 mM. The corresponding Vmax values were 418±12 and 354±13 nmol/min per mg of protein, for unlabelled and MIANS-labelled MsbA respectively. We previously reported that purified MsbA is homodimeric in DM solution [14]. When the kinetic data for wild-type and MIANS-labelled MsbA were fitted to the Hill equation, coefficients close to 1 were obtained (Figure 2B), suggesting that the two monomers function independently, and do not interact co-operatively with each other. This is consistent with the “alternating sites” proposal of Senior et al. [34] for the mechanism of Pgp, which is also proposed to be a dual drug transporter and lipid flippase; only one NBD is thought to be active at any instant in time, and the two sites alternate in catalysis.

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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