Function and inhibition of the lipopolysaccharide floppase MsbA

Abstract

MsbA is an ATP-binding Cassette (ABC) transporter tasked with flipping lipopolysaccharide (LPS) across the inner membrane of Gram-negative bacteria. Interrupting LPS transport by blocking MsbA leads to cell death, making MsbA an attractive target for developing new antibiotics against multidrug-resistant infections. Although high-resolution structural information can often accelerate drug discovery, studies of MsbA have thus far been complicated by the extraordinary conformational heterogeneity of this transporter. This dissertation exploits recent progress in cryo-electron microscopy (cryo-EM) to understand the MsbA function cycle and possible inhibition mechanisms. Imaging of MsbA in presence of different nucleotides shows that nucleotide binding is sufficient for substrate release and provides information about which conformations are achievable in lipid membranes and the relative prevalence of each state during continuous ATP turnover. Characterization of MsbA in complex with various modulators illustrates distinct mechanisms of inhibition by small molecules. Notably, the structure of MsbA bound to a decoupler of ATPase activity from LPS transport reveals that the substrate-binding pocket can be remodeled by induced-fit, and that it is an important site of allosteric regulation. In this novel inhibited ABC transporter conformation, significant rearrangements in the transmembrane domains lead to unproductive hydrolysis of ATP. In contrast, structures in complex with an ATPase suppressor demonstrates an inhibition mechanism involving symmetrical separation of the nucleotide-binding domains and impairment of the normal MsbA conformational transition. The findings presented here will inform the future development of modulators of MsbA and other ABC transporters.