title: Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.
abstract: The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherman
authors.forename: Michael B
authors.initials: MB
authors.lastname: Lazarus
authors.forename: Lea
authors.initials: L
authors.lastname: Murphy
authors.forename: Charles
authors.initials: C
authors.lastname: Liu
authors.forename: Suzanne
authors.initials: S
authors.lastname: Walker
authors.forename: Natividad
authors.initials: N
authors.lastname: Ruiz
authors.forename: Daniel
authors.initials: D
authors.lastname: Kahne
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D001692:Biological Transport; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008070:Lipopolysaccharides / Q000378:metabolism*; D050296:Microbial Viability; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; C543317:LptB protein, E coli; D050505:Mutant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: ABC transporter; antibiotic target; membrane biogenesis