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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
7.5.2.6 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
7.5.2.6 purified enzyme MsbA in complex with inhibitor G907, analysis of structure PDB ID 3B60 Salmonella enterica subsp. enterica serovar Typhimurium

Protein Variants

EC Number Protein Variants Comment Organism
7.5.2.6 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

EC Number Inhibitors Comment Organism Structure
7.5.2.6 (2E)-3-[1-cyclopropyl-7-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]naphthalen-2-yl]prop-2-enoic acid
-
Escherichia coli
7.5.2.6 (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
7.5.2.6 (2E)-3-[6-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid
-
Escherichia coli
7.5.2.6 (2E)-3-[6-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid
-
Salmonella enterica subsp. enterica serovar Typhimurium
7.5.2.6 (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
7.5.2.6 (2E)-3-[6-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]quinolin-3-yl]prop-2-enoic acid
-
Salmonella enterica subsp. enterica serovar Typhimurium
7.5.2.6 (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
7.5.2.6 (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
7.5.2.6 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
7.5.2.6 additional information analysis of ABC transporter inhibition mechanism, small molecule inhibitor library screening, overview Salmonella enterica subsp. enterica serovar Typhimurium

Localization

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

Metals/Ions

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

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
7.5.2.6 ATP + H2O + lipid A-core oligosaccharide[side 1] Salmonella enterica subsp. enterica serovar Typhimurium
-
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
?
7.5.2.6 ATP + H2O + lipid A-core oligosaccharide[side 1] Escherichia coli
-
ADP + phosphate + lipid A-core oligosaccharide[side 2]
-
?

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
7.5.2.6 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)

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

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
7.5.2.6 37
-
assay at Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5.2.6 7.5
-
assay at Escherichia coli

Cofactor

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

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
7.5.2.6 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
7.5.2.6 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

EC Number General Information Comment Organism
7.5.2.6 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
7.5.2.6 additional information inward facing structure of EcMsbA in complex with LPS, structure comparisons and molecular basis of active lipid transport, overview Escherichia coli
7.5.2.6 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
7.5.2.6 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
7.5.2.6 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