The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport

Abstract

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

Structural basis of unidirectional export of lipopolysaccharide to the cell surface

“…Protein samples with the tag cleaved were concentrated to 14 mg/ml, supplemented with 2 mM Na-novobiocin, and mixed at a 2:1 protein : precipitant ratio with a precipitant solution consisting of 500 mM Li 2 SO 4 , 100 mM MES (pH = 6.5), and 21% v/v PEG400. We had previously shown novobiocin binds to E. coli LptB 2 FGC and solved a cocrystal structure of LptB alone bound to novobiocin 27 , and so thought that it might stabilize a conformation of the full complex. In sparse-matrix screening with E. cloacae LptB 2 FGC we found several conditions which were dependent on the presence of Na-novobiocin in the protein sample.…”

“…Data were integrated in XDS 28 and scaled in the CCP4 29 suite program AIMLESS 30 . Molecular replacement for the E. cloacae data was performed in Phaser 31 using the transmembrane domains of K. pneumoniae LptB 2 FG (PDB ID 5L75) 19 , two copies of E. coli LptB (PDB ID 6B89) 27 and one copy of E. coli LptC (PDB ID 3MY2) 18 . After one round of refinement in Phenix 32 , transmembrane helix of LptC as well as most of the periplasmic domains of LptF and LptG were built manually in Coot 33 using 5L75 and 5X5Y to guide assignment of amino acid identity.…”

“…Mutant Construction.-Plasmids encoding LptF variants were generated by site-directed mutagenesis PCR using the pBAD18LptFG3 plasmid 36 as template, Phusion polymerase (Fischer Scientific), and the primers listed in Supplementary Table 1 (Sigma-Aldrich). Complementation testing and generation of haploid strains was carried out as described previously 27,36 .…”

Constructing and deconstructing the bacterial cell wall

“…Intimate structural synergy is now recognized between the peptidoglycan and the teichoic acids of the Gram‐positive bacteria, and between the peptidoglycan and the outer membrane of the Gram‐negative bacteria . Indeed, new opportunities for antibiotic discovery have been identified that interfere with teichoic acid integration into the cell wall of Gram‐positive bacteria and with respect to lipopolysaccharide transport in Gram‐negative bacteria . Within this universe of opportunity, we exemplify our own efforts toward the mechanistic and structural understanding of key enzymes of the bacteria with roles in cell envelope biosynthesis and antibiotic resistance.…”

“…77,78 Indeed, new opportunities for antibiotic discovery have been identified that interfere with teichoic acid integration into the cell wall of Gram-positive bacteria 69,[79][80][81][82][83][84] and with respect to lipopolysaccharide transport in Gram-negative bacteria. [85][86][87][88][89][90] Within this universe of opportunity, we exemplify our own efforts toward the mechanistic and structural understanding of key enzymes of the bacteria with roles in cell envelope biosynthesis and antibiotic resistance.…”