Title: Biochemical and Biophysical Characterization of the Dysfunctional E506Q MsbA Protein
Abstract: MsbA is a 65kDa ABC transporter found in the inner membrane of Gram-negative bacteria. The ABC transporter superfamily is one of the largest known and is responsible for the transport of a variety of substrates from lipids to antibiotics. ABC transporter dysfunction is involved in a range of human pathologies from cystic fibrosis to Stargardt's macular dystrophy. MsbA functions as a homodimer comprised of two nucleotide binding domains and two transmembrane domains and transports lipid A across the inner membrane of Gram-negative bacteria. MsbA is an essential protein in E. coli and its deletion or dysfunction results in the accumulation of lipid A in the inner membrane causing membrane instability and cell death. The E506Q mutation in the nucleotide binding domain is dysfunctional in MsbA and is a well-known mutation within the ABC transporter superfamily. To characterize this dysfunctional mutation for the first time in MsbA, in vivo growth assays, in vitro ATPase activity assays, and EPR spectroscopy studies throughout the ATP hydrolysis cycle were conducted. The E506Q mutation was paired with nine different reporter residues, each in or near a key nucleotide binding domain motif. Each pair has been characterized by site-directed spin labeling, ATPase assays, and an in vivo growth assay and compared to the reporter residues alone. To identify the stage in the ATP hydrolysis cycle in which the E506Q mutation is dysfunctional, the local tertiary interactions before, during, and after ATP hydrolysis were monitored by EPR spectroscopy at each stage of the ATP hydrolysis cycle, using ATP, ADP, and ATP and vanadate. With the ATPase activity, growth rate, and EPR spectroscopy compared between the reporters alone and paired with the E506Q mutation, the functional step at which the E506Q mutation dysfunctions has been identified in MsbA.