title: Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.
abstract: In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.
authors.affiliation: Discovery Sciences, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA. Electronic address: ning.gao@astrazeneca.com.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: Department of Bioscience, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: McLeod
authors.affiliation: Department of Bioscience, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Laurel
authors.initials: L
authors.lastname: Hajec
authors.affiliation: Discovery Sciences, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Olivier
authors.affiliation: Department of Bioscience, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Sushmita D
authors.initials: SD
authors.lastname: Lahiri
authors.affiliation: Discovery Sciences, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: D
authors.initials: D
authors.lastname: Bryan Prince
authors.affiliation: Department of Bioscience, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
authors.affiliation: Discovery Sciences, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Philip L
authors.initials: PL
authors.lastname: Ross
authors.affiliation: Department of Bioscience, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: James D
authors.initials: JD
authors.lastname: Whiteaker
authors.affiliation: Discovery Sciences, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Doig
authors.affiliation: Department of Chemistry, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Amanda Haixi
authors.initials: AH
authors.lastname: Li
authors.affiliation: Department of Chemistry, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Hill
authors.affiliation: Department of Chemistry, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Cornebise
authors.affiliation: Department of Chemistry, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Folkert
authors.initials: F
authors.lastname: Reck
authors.affiliation: Department of Chemistry, Infection Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hale
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics*; D015870:Gene Expression; D013058:Mass Spectrometry; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D000581:Amidohydrolases; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015032:Zinc; C083496:AcrB protein, E coli; C514484:LpxC deacetylase, Pseudomonas
keywords: Crystallization; Inhibitor; LpxC; Protein expression; Protein purification; acrB