Literature summary for 7.5.2.6 extracted from

Ho, H.; Miu, A.; Alexander, M.K.; Garcia, N.K.; Oh, A.; Zilberleyb, I.; Reichelt, M.; Austin, C.D.; Tam, C.; Shriver, S.; Hu, H.; Labadie, S.S.; Liang, J.; Wang, L.; Wang, J.; Lu, Y.; Purkey, H.E.; Quinn, J.; Franke, Y.; Clark, K.; Beresini, M.H.; Tan, M.W.; Sellers, B.D.; Maurer, T.; Koehler, M.F.T.; Wecksler, A.T.
Structural basis for dual-mode inhibition of the ABC transporter MsbA (2018), Nature, 557, 196-201 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene msbA	Salmonella enterica subsp. enterica serovar Typhimurium
gene msbA, recombinant overexpresssion of FLAG-tagged enzyme in Escherichia coli strain Rosetta 2 (DE3)	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme MsbA free and in complex with inhibitors (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid or G902, sitting drop vapour diffusion method, mixing of 10 mg/ml protein in 50 mM Tris, pH 8.0, 500 mM NaCl, and 0.6% FA-3 detergent, with mother liquor containing 200 mM NaCl, 19% PEG 550 MME, 4% PEG 400, and 100 mM HEPES, pH 7.0, in a 1:1 v/v ratio, 19°C, 1 week, X-ray diffraction structure determination and analysis at 2.9 A resolution	Escherichia coli
purified enzyme MsbA in complex with inhibitor G907, analysis of structure PDB ID 3B60	Salmonella enterica subsp. enterica serovar Typhimurium

Crystallization (Comment)

Organism

purified enzyme MsbA free and in complex with inhibitors (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid or G902, sitting drop vapour diffusion method, mixing of 10 mg/ml protein in 50 mM Tris, pH 8.0, 500 mM NaCl, and 0.6% FA-3 detergent, with mother liquor containing 200 mM NaCl, 19% PEG 550 MME, 4% PEG 400, and 100 mM HEPES, pH 7.0, in a 1:1 v/v ratio, 19°C, 1 week, X-ray diffraction structure determination and analysis at 2.9 A resolution

Escherichia coli

purified enzyme MsbA in complex with inhibitor G907, analysis of structure PDB ID 3B60

Salmonella enterica subsp. enterica serovar Typhimurium

Protein Variants

Protein Variants	Comment	Organism
additional information	overexpression of msbA suppresses mutations in the htrB lipid A acyltransferase, as MsbA can transport the tetra-acylated LPS produced in htrB mutants, albeit inefficiently	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism
(2E)-3-[1-cyclopropyl-7-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]naphthalen-2-yl]prop-2-enoic acid	-	Escherichia coli
(2E)-3-[1-cyclopropyl-7-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]naphthalen-2-yl]prop-2-enoic acid	-	Salmonella enterica subsp. enterica serovar Typhimurium
(2E)-3-[6-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid	-	Escherichia coli
(2E)-3-[6-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid	-	Salmonella enterica subsp. enterica serovar Typhimurium
(2E)-3-[6-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid	a quinoline compound with potent activity on purified Escherichia coli MsbA	Escherichia coli
(2E)-3-[6-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid	-	Salmonella enterica subsp. enterica serovar Typhimurium
(2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid	selective small-molecule antagonist with bactericidal activity and a dual-mode inhibitory mechanism. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid gains access to MsbA through the bulk membrane. The 2-chloro-6 cyclopropylphenyl substituent of (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid (A-ring) exhibits strong electron density and makes van der Waals interactions with side chains from transmembrane domains TM4 (L171, A175 and V178), TM5 (A259 and L263) and TM6 (M291 and L294). The quinoline core of (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid (B-ring) is orthogonal to the plane of the phenyl substituent, where it is partially enclosed by residues from transmembrane domains TM4 (V178, S179 and I182), TM5 (A259) and TM6 (M295 and L298). A259 and M295 side chains contact the 4-cyclopropyl substitution of the quinoline core and delineate the inhibitor binding-pocket from the inner vestibule of MsbA. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid stabilizes a catalytically incompetent state of the transporter. The inhibitor may have broad relevance across the ABC transporter superfamily	Escherichia coli
(2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid	i.e. G907, selective small-molecule antagonist with bactericidal activity and a dual-mode inhibitory mechanism. G907-MsbA binding structure, overview. In the outward-facing StMsbA structure, the G907 pocket is markedly deformed and expected to perturb inhibitor binding. This substantial perturbation of the binding site during the transport cycle defines G907 as an inward-facing state-dependent inhibitor. The inhibitor may have broad relevance across the ABC transporter superfamily	Salmonella enterica subsp. enterica serovar Typhimurium
additional information	analysis of ABC transporter inhibition mechanism, small molecule inhibitor library screening, overview. (2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains	Escherichia coli
additional information	analysis of ABC transporter inhibition mechanism, small molecule inhibitor library screening, overview	Salmonella enterica subsp. enterica serovar Typhimurium

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
periplasm	-	Salmonella enterica subsp. enterica serovar Typhimurium	-	-
periplasm	-	Escherichia coli	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Salmonella enterica subsp. enterica serovar Typhimurium
Mg2+	required	Escherichia coli

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]	Salmonella enterica subsp. enterica serovar Typhimurium	-	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?
ATP + H2O + lipid A-core oligosaccharide[side 1]	Escherichia coli	-	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P60752	-	-
Salmonella enterica subsp. enterica serovar Typhimurium	P63359	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant FLAG-tagged enzyme from Escherichia coli strain Rosetta 2 (DE3) by affinity chromatography, the Flag tag is removed using TEV protease, followed by gel filtration and ultrafiltration	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + H2O + lipid A-core oligosaccharide[side 1]	-	Salmonella enterica subsp. enterica serovar Typhimurium	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?
ATP + H2O + lipid A-core oligosaccharide[side 1]	-	Escherichia coli	ADP + phosphate + lipid A-core oligosaccharide[side 2]	-	?
additional information	MsbA cannot efficiently transport a substrate lacking phosphorylation at the 4'-position of lipid A	Salmonella enterica subsp. enterica serovar Typhimurium	?	-	?
additional information	MsbA cannot efficiently transport a substrate lacking phosphorylation at the 4'-position of lipid A	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
EcMsbA	-	Escherichia coli
lipid A export ATP-binding/permease protein MsbA	UniProt	Escherichia coli
MsbA	-	Salmonella enterica subsp. enterica serovar Typhimurium
MsbA	-	Escherichia coli
STM0984	locus name	Salmonella enterica subsp. enterica serovar Typhimurium
StMsbA	-	Salmonella enterica subsp. enterica serovar Typhimurium

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Salmonella enterica subsp. enterica serovar Typhimurium
ATP	-	Escherichia coli

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.000005	-	pH 7.5, 37°C, recombinant detagged enzyme	Escherichia coli	(2E)-3-[1-cyclopropyl-7-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]naphthalen-2-yl]prop-2-enoic acid
0.000018	-	pH 7.5, 37°C, recombinant detagged enzyme	Escherichia coli	(2E)-3-[6-[1-(2-chloro-6-cyclopropylphenyl)ethoxy]-4-cyclopropylquinolin-3-yl]prop-2-enoic acid

General Information

General Information	Comment	Organism
metabolism	the enzyme is involved in the lipid A biosynthesis. MsbA cannot efficiently transport a substrate lacking phosphorylation at the 4'-position of lipid A. This last essential step of lipid A biosynthesis is catalysed by LpxK, the two proteins are concatenated in some bacteria, suggesting tight coupling of their cellular activities	Escherichia coli
additional information	inward facing structure of EcMsbA in complex with LPS, structure comparisons and molecular basis of active lipid transport, overview	Escherichia coli
additional information	outward facing structure of Salmonella typhimurium MsbA (StMsbA) in complex with LPS, structure comparisons and molecular basis of active lipid transport, overview	Salmonella enterica subsp. enterica serovar Typhimurium
physiological function	the movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA, structure of LPS in complex with EcMsbA, overview	Escherichia coli
physiological function	the movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA, structure of LPS in complex with StMsbA, overview	Salmonella enterica subsp. enterica serovar Typhimurium