title: Functional characterization of E. coli LptC: interaction with LPS and a synthetic ligand.
abstract: Lipopolysaccharide (LPS), the main cell-surface molecular constituent of Gram-negative bacteria, is synthesized in the inner membrane (IM) and transported to the outer membrane (OM) by the Lpt (lipopolysaccharide transport) machinery. Neosynthesized LPS is first flipped by MsbA across the IM, then transported to the OM by seven Lpt proteins located in the IM (LptBCFG), in the periplasm (LptA), and in the OM (LptDE). A functional OM is essential to bacterial viability and requires correct placement of LPS in the outer leaflet. Therefore, LPS biogenesis represents an ideal target for the development of novel antibiotics against Gram-negative bacteria. Although the structures of Lpt proteins have been elucidated, little is known about the mechanism of LPS transport, and few data are available on Lpt–LPS binding. We report here the first determination of the thermodynamic and kinetic parameters of the interaction between LptC and a fluorescent lipo-oligosaccharide (fLOS) in vitro. The apparent dissociation constant (Kd) of the fLOS–LptC interaction was evaluated by two independent methods. The first was based on fLOS capture by resin-immobilized LptC; the second used quenching of LptC intrinsic fluorescence by fLOS in solution. The Kd values by the two methods (71.4 and 28.8 μm, respectively) are very similar, and are of the same order of magnitude as that of the affinity of LOS for the upstream transporter, MsbA. Interestingly, both methods showed that fLOS binding to LptC is mostly irreversible, thus reflecting the fact that LPS can be released from LptC only when energy is supplied by ATP or in the presence of a higher-affinity LptA protein. A fluorescent glycolipid was synthesized: this also interacted irreversibly with LptC, but with lower affinity (apparent Kd=221 μM). This compound binds LptC at the LPS binding site and is a prototype for the development of new antibiotics targeting LPS transport in Gram-negative bacteria.
authors.forename: Stefania E
authors.initials: SE
authors.lastname: Sestito
authors.forename: Paola
authors.initials: P
authors.lastname: Sperandeo
authors.forename: Carlo
authors.initials: C
authors.lastname: Santambrogio
authors.forename: Carlotta
authors.initials: C
authors.lastname: Ciaramelli
authors.forename: Valentina
authors.initials: V
authors.lastname: Calabrese
authors.forename: G Enrico
authors.initials: GE
authors.lastname: Rovati
authors.forename: Luca
authors.initials: L
authors.lastname: Zambelloni
authors.forename: Rita
authors.initials: R
authors.lastname: Grandori
authors.forename: Alessandra
authors.initials: A
authors.lastname: Polissi
authors.forename: Francesco
authors.initials: F
authors.lastname: Peri
mesh_terms: D001692:Biological Transport; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; C543318:LptC protein, E coli; D008565:Membrane Proteins; D009844:Oligosaccharides