Structural and Biochemical Characterization of the ABC-Transporter That Powers Lipopolysaccharide Transport in Gram-Negative Bacteria

Abstract

The cell envelope of Gram-negative bacteria has a unique double-membrane architecture that limits the entry of antibiotics and allows them to colonize harsh environments. The cytoplasmic membrane and peptidoglycan cell wall, typical of all bacteria, are surrounded by an outer membrane with an outer leaflet composed primarily of lipopolysaccharide (LPS) rather than phospholipids. LPS is composed of hundreds of sugars and several saturated acyl chains, and the asymmetric distribution of LPS in the outer membrane makes Gram-negatives unusually impermeable to hydrophobic compounds. The cytoplasmic and inner-membrane enzymes involved in the biosynthesis of LPS have been studied in great detail, however, the proteins responsible for its assembly in the outer membrane have only been identified recently. In E. coli, seven essential LPS transport proteins, LptA-LptG, transport LPS from the outer leaflet of the inner membrane, across the aqueous periplasm, to the outer leaflet of the outer membrane. The cytoplasmic and inner membrane Lpt components form complex, LptB2FGC, which is a member of the ATP-binding-cassette (ABC) -transporter superfamily and uses the energy released in ATP binding and hydrolysis to extract LPS from the inner membrane and power its transport to the outer membrane. The mechanism by which cytoplasmic LptB couples ATP cycling to LPS extraction from the membrane remains mysterious, as do the means by which LptB2FGC ensures unidirectional LPS transport to the outer membrane. In the first part of this thesis, we use structural, biochemical, and genetic tools to interrogate the interactions between LptB and LptFG. We find that although LptB2FGC is an atypical ABC-transporter, it contains several motifs common to other members of the family and uses widely conserved interactions between the nucleotide binding domains and transmembrane domains to mediate LPS transport. During these studies, we make the serendipitous discovery that the antibiotic novobiocin binds to Lpt and is an agonist of LPS transport. As LPS is both essential in most Gram-negative bacteria and a primary factor in their antibiotic resistance, structural and mechanistic characterization of the Lpt proteins could enable the development of new antibiotics. In the second part of this thesis we report the first X-ray crystal structures of LptB2FGC and use information glean from them to guide functional studies of LPS transport. We identify the path LPS takes out of the membrane, into a cavity within the transporter, and on to the Lpt bridge to the outer membrane. We find that LPS entry into the cavity is ATP-independent whereas extraction from the membrane require ATP. Comparison of the structures reveals a protein gate that must open to allow LPS movement out of the cavity and onto the bridge. Together, our findings show how LptB2FGC acheives efficient unidirectional transport of LPS to the outer membrane.