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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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CD spectroscopy of MsbA and MsbA–MIANS(A) CD spectra of unlabelled MsbA (continuous line) and MsbA–MIANS (dotted line) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. (B) Thermal unfolding of MsbA and MsbA–MIANS monitored by CD spectroscopy. CD measurements were carried out on purified unlabelled MsbA (●) and MsbA–MIANS (○) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. Molar ellipticity was recorded at 222 nm, which reports on α-helical unfolding.
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Figure 5: CD spectroscopy of MsbA and MsbA–MIANS(A) CD spectra of unlabelled MsbA (continuous line) and MsbA–MIANS (dotted line) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. (B) Thermal unfolding of MsbA and MsbA–MIANS monitored by CD spectroscopy. CD measurements were carried out on purified unlabelled MsbA (●) and MsbA–MIANS (○) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. Molar ellipticity was recorded at 222 nm, which reports on α-helical unfolding.

Mentions: The CD spectra of native MsbA and MsbA–MIANS were essentially superimposable (Figure 5A), indicating that covalent linkage of MIANS does not perturb the overall protein secondary structure. Analysis of the CD spectrum using the DICHROWEB server [36] indicated that MsbA comprises 58% α-helix, 9% β-strand and 34% random coil/unassigned structure (8% maximum error). These data are in good agreement with X-ray structures reported for MsbA from three bacterial species [15], where α-helices make up a large proportion of the secondary structure (63% α-helix, 10% β-strand; http://www.rcsb.org). The conformational stability of proteins can be assessed by thermal or chemical denaturation, which can be monitored by CD spectroscopy [37]. Thermal denaturation of native and MIANS-labelled MsbA resulted in CD spectral changes from 195–275 nm indicative of a coincident decrease in both secondary and tertiary structure. Figure 5(B) shows the temperature-induced CD change in MsbA at 222 nm, which was chosen because ellipticity changes at this wavelength reflect alterations in α-helical content [38]. The thermally-induced changes in secondary structure for both native and MIANS-labelled MsbA are essentially identical, showing that they unfold to the same extent, and with the same rate and temperature dependence. Thus, the folding and stability of MsbA is essentially unaltered after covalent attachment of MIANS.


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

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

CD spectroscopy of MsbA and MsbA–MIANS(A) CD spectra of unlabelled MsbA (continuous line) and MsbA–MIANS (dotted line) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. (B) Thermal unfolding of MsbA and MsbA–MIANS monitored by CD spectroscopy. CD measurements were carried out on purified unlabelled MsbA (●) and MsbA–MIANS (○) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. Molar ellipticity was recorded at 222 nm, which reports on α-helical unfolding.
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Figure 5: CD spectroscopy of MsbA and MsbA–MIANS(A) CD spectra of unlabelled MsbA (continuous line) and MsbA–MIANS (dotted line) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. (B) Thermal unfolding of MsbA and MsbA–MIANS monitored by CD spectroscopy. CD measurements were carried out on purified unlabelled MsbA (●) and MsbA–MIANS (○) at a concentration of 0.35 mg/ml in buffer with 0.01% (w/v) DM. Molar ellipticity was recorded at 222 nm, which reports on α-helical unfolding.
Mentions: The CD spectra of native MsbA and MsbA–MIANS were essentially superimposable (Figure 5A), indicating that covalent linkage of MIANS does not perturb the overall protein secondary structure. Analysis of the CD spectrum using the DICHROWEB server [36] indicated that MsbA comprises 58% α-helix, 9% β-strand and 34% random coil/unassigned structure (8% maximum error). These data are in good agreement with X-ray structures reported for MsbA from three bacterial species [15], where α-helices make up a large proportion of the secondary structure (63% α-helix, 10% β-strand; http://www.rcsb.org). The conformational stability of proteins can be assessed by thermal or chemical denaturation, which can be monitored by CD spectroscopy [37]. Thermal denaturation of native and MIANS-labelled MsbA resulted in CD spectral changes from 195–275 nm indicative of a coincident decrease in both secondary and tertiary structure. Figure 5(B) shows the temperature-induced CD change in MsbA at 222 nm, which was chosen because ellipticity changes at this wavelength reflect alterations in α-helical content [38]. The thermally-induced changes in secondary structure for both native and MIANS-labelled MsbA are essentially identical, showing that they unfold to the same extent, and with the same rate and temperature dependence. Thus, the folding and stability of MsbA is essentially unaltered after covalent attachment of MIANS.

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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