Literature summary for 7.5.2.5 extracted from

Sherman, D.J.; Lazarus, M.B.; Murphy, L.; Liu, C.; Walker, S.; Ruiz, N.; Kahne, D.
Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport (2014), Proc. Natl. Acad. Sci. USA, 111, 4982-4987 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene lptB, recombinant expression of His8-tagged wild-type and mutant LptB enzymes and of His6-tagged LptB-LptFG in Escherichia coli strain DH5alpha and in Escherichia coli KRX cells from different plasmid constructions, recombinant expression of selenomethionine-labeled enzyme from Escherichia coli strain BL21(DE3)	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystal structures of recombinant His-tagged LptB pre- and post-ATP hydrolysis, for native enzyme: mixing of 0.001 ml of 7-10 mg/mL protein in 10% v/v glycerol, 2.5 mM ATP, 2.5 mM MgCl2, 150 mM NaCl, and 20 mM Tris, pH 8.0, with 0.001 ml of reservoir solution containing 0.1 M MES, pH 6.5, and 30% w/v PEG 4000, and for SeMet-LptB-His enzyme: mixing of 0.002 ml of 7-10 mg/mL protein in 10% v/v glycerol, 2.5 mM ATP, 2.5 mM MgCl2, 150 mM NaCl, and 20 mM Tris, pH 8.0, with 0.001 ml of reservoir solution composed of 0.1 M MES, pH 6.5, and 31% w/v PEG 4000, room temperature, several days, X-ray diffraction structure determination and analysis, molecular replacement using the native LptB-Mg2+-ADP structure as the search model	Escherichia coli

Crystallization (Comment)

Organism

crystal structures of recombinant His-tagged LptB pre- and post-ATP hydrolysis, for native enzyme: mixing of 0.001 ml of 7-10 mg/mL protein in 10% v/v glycerol, 2.5 mM ATP, 2.5 mM MgCl2, 150 mM NaCl, and 20 mM Tris, pH 8.0, with 0.001 ml of reservoir solution containing 0.1 M MES, pH 6.5, and 30% w/v PEG 4000, and for SeMet-LptB-His enzyme: mixing of 0.002 ml of 7-10 mg/mL protein in 10% v/v glycerol, 2.5 mM ATP, 2.5 mM MgCl2, 150 mM NaCl, and 20 mM Tris, pH 8.0, with 0.001 ml of reservoir solution composed of 0.1 M MES, pH 6.5, and 31% w/v PEG 4000, room temperature, several days, X-ray diffraction structure determination and analysis, molecular replacement using the native LptB-Mg2+-ADP structure as the search model

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
E163Q	site-directed mutagenesis, catalytically inactive variant, introduction of plasmid-encoded LptBE163Q-His8 variant into the wild-type strain results in increased outer membrane permeability, dominant-negative phenotype	Escherichia coli
F90A	site-directed mutagenesis, catalytically inactive variant, the F90A variant cannot form a stable complex with the Lpt-inner membrane components	Escherichia coli
F90Y	site-directed mutagenesis, the LptB and LptB-His8 F90Y variants are functional in vivo	Escherichia coli
H195A	site-directed mutagenesis, catalytically inactive variant, introduction of plasmid-encoded LptB-H195A-His8 variant into the wild-type strain results in increased outer membrane permeability, dominant-negative phenotype	Escherichia coli
additional information	construction of chromosomal deletion of lptB and replacement with a kanamycin-resistance cassette (DELTAlptB:: kan allele), where the region from the second codon to the stop codon of lptB is deleted	Escherichia coli

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
inner membrane	as LptB2FGC enzyme complex, residue F90 is essential for proper formation of the Lpt inner membrane complex, transmembrane complex. The groove region of LptB is essential for interaction with inner membrane partners	Escherichia coli	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A9V1	-	-

Purification (Commentary)

Purification (Comment)	Organism
active recombinant His8-tagged wild-type and mutant LptB enzymes and His6-tagged LptB-LptFG from Escherichia coli strains for activity assays and crystallization are purified by ultracentrifugation, nickel affinity chromatography, ultrafiltration, and gel filtration, recombinant His6/8-tagged enzyme for immunoblotting is purified from Escherichia coli by ultracentrifugation, nickel affinity chromatography, trichloroacetate precipitation and acetone wash, boiling, and SDS-PAGE	Escherichia coli

Purification (Comment)

Organism

active recombinant His8-tagged wild-type and mutant LptB enzymes and His6-tagged LptB-LptFG from Escherichia coli strains for activity assays and crystallization are purified by ultracentrifugation, nickel affinity chromatography, ultrafiltration, and gel filtration, recombinant His6/8-tagged enzyme for immunoblotting is purified from Escherichia coli by ultracentrifugation, nickel affinity chromatography, trichloroacetate precipitation and acetone wash, boiling, and SDS-PAGE

Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	ATPase assays are conducted for both LptB and LptB2FGC variants	Escherichia coli	?	-	?

Subunits

Subunits	Comment	Organism
More	LptB possesses an overall fold resembling that of nucleoside triphosphate-binding (NBD) structures. It contains the canonical L-shaped architecture composed of a RecA-like alpha/beta-ATPase domain and a structurally diverse alpha-helical domain. The RecA-like domain contains the Walker A and Walker B motifs present in many NBD proteins. This domain also furnishes Mg2+- and nucleotide-binding motifs specific to ABC proteins, namely, the Q-loop, which links the more highly conserved alpha/beta-ATPase domain to the alpha-helical domain, and the switch region, which contains the conserved H195	Escherichia coli

Synonyms

Synonyms	Comment	Organism
lptB	-	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	ATP hydrolysis induces conformational changes, the switch region moves and the side chain of H195 flips, ATP binding structure, overview. The H195 is imidazole side chain directly interacts with the gamma-phosphate of ATP. Through a bridging water molecule, the glutamate E163 contacts the beta-phosphate of the nucleotide	Escherichia coli

General Information

General Information	Comment	Organism
malfunction	mutations in the nucleotide-binding domain, LptB, of the transporter inactivates transporter function in vivo	Escherichia coli
additional information	residue F90 is essential for proper formation of the Lpt inner membrane complex. crystal structures of LptB pre- and post-ATP hydrolysis suggest a role for an active site residue in phosphate exit. Residues E163, H195, and F90 of LptB are required for cell viability. E163 is essential for catalysis, through a bridging water molecule, this glutamate contacts the beta-phosphate of the nucleotide. ATP hydrolysis induces conformational changes. Conformational changes upon ATP hydrolysis show how reorganization of the active site causes changes in the region of LptB believed to interact with LptF/G. The dramatic movement of the switch region observed during ATP hydrolysis plays a critical role in communicating changes in the active site to changes in the transmembrane domains. The groove region of LptB is essential for interaction with inner membrane partners	Escherichia coli