Literature summary for 7.5.2.6 extracted from

Singh, H.; Velamakanni, S.; Deery, M.; Howard, J.; Wei, S.; Van Veen, H.
ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled (2016), Nat. Commun., 7, 12387 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene msbA, recombinant expression of N-terminally His-tagged wild-type and mutant MsbAs in Escherichia coli	Lactococcus lactis subsp. lactis

Protein Variants

Protein Variants	Comment	Organism
D41N/E149Q/D252N	site-directed mutagenesis, triple mutant MsbA-DED, comprising mutations D41N in transmembrane helix (TMH) 1, E149Q in TMH 3 and D252N in TMH 5, is transport inactive	Lactococcus lactis subsp. lactis
additional information	construction of a truncated mutant form MsbA-MD of wild-type MsbA that lacks the nucleotide-binding domain (NBD). Truncated mutant MsbA-DELTAK382 exhibits a strongly reduced ATPase activity due to the absence of the catalytic Walker A lysine residue. Preparation of proteoliposomes from Escherichia coli phospholipids demonstrating the equal incorporation of purified transport-inactive triple mutant MsbA-DED, truncated mutant MsbA-MD, mutant MsbA-DELTAK382 and wild-type MsbA, and the absence of membrane proteins in empty control liposomes. Observations on active drug transport by MsbA-MD and wild-type MsbA show that wild-type MsbA is more efficient than MsbA-MD in vivo	Lactococcus lactis subsp. lactis

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
inner membrane	transmembrane enzyme	Lactococcus lactis subsp. lactis	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Lactococcus lactis subsp. lactis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + H2O + lipid A-core oligosaccharide[side 1]	Lactococcus lactis subsp. lactis	-	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?
ATP + H2O + lipid A-core oligosaccharide[side 1]	Lactococcus lactis subsp. lactis NZ9000	-	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?

Organism

Organism	UniProt	Comment	Textmining
Lactococcus lactis subsp. lactis	A0A2N5WD20	-	-
Lactococcus lactis subsp. lactis NZ9000	A0A2N5WD20	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His-tagged wild-type and mutant MsbAs from Escherichia coli, preparation of inside-out membrane vesicles, reconstitution of purified MsbA proteins in proteoliposomes, solubilization by 1% w/v n-dodecyl-beta-D-maltoside, and further purification by nickel affinity chromatography. The orientation of MsbA proteins in right-side-out membrane vesicles, inside-out membrane vesicles or proteoliposomes is assessed by determining the accessibility of the N-terminal His-tag to digestion by protease K in the external buffer	Lactococcus lactis subsp. lactis

Purification (Comment)

Organism

recombinant N-terminally His-tagged wild-type and mutant MsbAs from Escherichia coli, preparation of inside-out membrane vesicles, reconstitution of purified MsbA proteins in proteoliposomes, solubilization by 1% w/v n-dodecyl-beta-D-maltoside, and further purification by nickel affinity chromatography. The orientation of MsbA proteins in right-side-out membrane vesicles, inside-out membrane vesicles or proteoliposomes is assessed by determining the accessibility of the N-terminal His-tag to digestion by protease K in the external buffer

Lactococcus lactis subsp. lactis

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + H2O + lipid A-core oligosaccharide[side 1] = ADP + phosphate + lipid A-core oligosaccharide[side 2]	ion coupling is a part of the mechanism of the homodimeric ABC transporter MsbA	Lactococcus lactis subsp. lactis	a

Renatured (Commentary)

Renatured (Comment)	Organism
reconstitution of purified His-tagged wild-type and mutant MsbA proteins in proteoliposomes	Lactococcus lactis subsp. lactis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + H2O + lipid A-core oligosaccharide[side 1]	-	Lactococcus lactis subsp. lactis	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?
ATP + H2O + lipid A-core oligosaccharide[side 1]	-	Lactococcus lactis subsp. lactis NZ9000	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?

Subunits

Subunits	Comment	Organism
homodimer	-	Lactococcus lactis subsp. lactis

Synonyms

Synonyms	Comment	Organism
Lipid A transporter	-	Lactococcus lactis subsp. lactis
MsbA	-	Lactococcus lactis subsp. lactis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Lactococcus lactis subsp. lactis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Lactococcus lactis subsp. lactis

Cofactor

Cofactor	Comment	Organism	Structure
ATP	the ATP dependence therefore enhances the directionality of the transport reaction	Lactococcus lactis subsp. lactis

General Information

General Information	Comment	Organism
additional information	preparation of proteoliposomes from Escherichia coli phospholipids demonstrating the equal incorporation of purified transport-inactive triple mutant MsbA-DED, truncated mutant MsbA-MD, mutant MsbA-DELTAK382 and wild-type MsbA, and the absence of membrane proteins in empty control liposomes. Proton-coupled substrate transport in proteoliposomes. The enhanced efficiency of efflux by full-length MsbA compared with the nucleotide-binding domain (NBD)-less protein is also found in the ability of the MsbA proteins to confer cellular resistance to the antibiotic erythromycin. The ATP-dependent dimerization of the NBDs with closure of the substrate-binding cavity towards the inside surface of the membrane facilitates capture of substrate from the cellular interior and/or inner membrane leaflet, and enables efflux against a larger drug concentration gradient and/or lipid-water partition coefficient. The ATP dependence therefore enhances the directionality of the transport reaction	Lactococcus lactis subsp. lactis
physiological function	ATP-binding cassette (ABC) multidrug exporters are embedded in the plasma membrane and actively extrude cytotoxic drugs from the cell. Alternating access models for ABC exporters including the multidrug and lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward and outward facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. MsbA catalyzes proton-coupled substrate transport in proteoliposomes. Energy coupling by MsbA, detailed overview	Lactococcus lactis subsp. lactis