Characterization of the L511P and D512G Mutations in the MsbA Lipid Flippase

Abstract

MsbA is a 65kDa lipid flippase found in the inner membrane of Gram-negative bacteria such as E. coli and S. typhimurium. As a member of the ABC transporter superfamily, MsbA contains two nucleotide binding domains and two transmembrane domains, one from each of its two monomers. ABC transporters transport a diverse group of substrates from lipids to antibiotics and their dysfunction contributes to a number of human pathologies including cystic fibrosis. As an essential protein in E. coli, the deletion or dysfunction of MsbA results in the toxic accumulation of lipid A in the inner membrane resulting in membrane instability and cell death. The L511P and D512G mutations have been previously identified through mutational analysis as dysfunctional nucleotide binding domain mutations specific to MsbA and were suggested to have a lower affinity for ATP. To further understand the cause of dysfunction in these point mutations, in vivo growth assays, in vitro ATPase activity assays, DEER and CW EPR spectroscopy studies throughout the ATP hydrolysis cycle were conducted. L511P and D512G were each paired with nine different reporter residues, each in or near an important conserved nucleotide binding domain motif and compared to the reporter residues alone. To identify the stage in the ATP hydrolysis cycle in which the L511P and D512G mutations are dysfunctional, the local tertiary interactions before, during, and after ATP hydrolysis were monitored by EPR spectroscopy at each stage of the ATP hydrolysis cycle.