title: Dimerization of isolated Pseudomonas aeruginosa lipopolysaccharide transporter component LptA.
abstract: LptA is a soluble periplasmic component of the lipopolysaccharide (LPS) transport system of Gram-negative bacteria that transports newly synthesized LPS from the inner membrane to the outer leaflet of the outer membrane. LptA links the inner membrane components (LptBFGC) to the outer membrane components (LptDE), but it is uncertain whether LptA is a freely moving LPS shuttle or part of a stable trans-periplasm structure. Escherichiacoli LptA forms highly polymerized head-to-tail oligomers in solution, but dimers in vivo. We studied the oligomerization of purified Pseudomonasaeruginosa LptA. Size-exclusion chromatography showed that P. aeruginosa LptA, unlike E. coli LptA, is a dimer over a wide range of concentrations. Chemical crosslinking with bis(sulfosuccinimidyl) suberate confirmed that dimers were the predominant species even at sub-micromolar LptA concentrations, which was unaffected by LPS binding. Mass spectrometry of crosslinked dimers showed that crosslinks occurred between the N-terminal α-amino group and either Lys-172 or Lys-173 near the C-terminus. These results support a hypothetical structure for the dimer of isolated P. aeruginosa LptA in which the N-terminus of one monomer is in close proximity to the C-terminus of the other, and the same surface of each monomer forms the interface between them, preventing further oligomerization.
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, United States. Electronic address: adam.shapiro@astrazeneca.com.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, United States.
authors.forename: Rong-Fang
authors.initials: RF
authors.lastname: Gu
authors.affiliation: Reagents & Assay Development, Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA, United States.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C532155:LptA protein, E coli
keywords: Crosslinking; LPS; Lipopolysaccharide; LptA; Transporter