Functionally Important ATP Binding and Hydrolysis Sites in Escherichia coli MsbA

Abstract

ATP-binding cassette (ABC) transporters make up one of the largest classes of proteins found in nature, and their ability to move a variety of substrates across the membrane using energy from the binding or hydrolysis of ATP is essential to an array of human pathologies and to bacterial viability. MsbA is an essential ABC transporter that specifically transports lipid A across the inner membranes of Gram-negative organisms such as Escherichia coli. The exact mechanisms of function during the binding and hydrolysis of ATP at the molecular level remain unclear. The studies presented and summarized in this work directly address the role and local dynamics of specific residues within the conserved ABC motifs in E. coli MsbA using in vivo growth and biochemical activity assays coupled with site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy motional and accessibility analysis. This first comprehensive analysis of the specific residues in these motifs within MsbA indicates that closure of the dimer interface does not occur upon ATP binding in this transporter.ATP-binding cassette (ABC) 1 transporters constitute one of the largest and most important families of integral membrane proteins. They specifically contribute to drug resistance by using ATP hydrolysis to export delivered drugs back across the cell membrane, and the function or dysfunction of other known ABC transporters results in serious genetic disorders such as cystic fibrosis (1). ABC transporters are typically comprised of two membrane-spanning domains and two intracellular ATP binding domains [or nucleotide binding domains (NBDs)].MsbA is an ABC transporter that functions as a homodimer with a molecular mass of 130 kDa and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Vibrio cholera, and Pseudomonas aeruginosa (2). Its role is to transport the negatively charged lipid A across the hydrophobic bacterial inner membrane. Because lipid A is the major component of the outer leaflet of the outer membrane of Gramnegative bacteria, its synthesis and transport are essential for cell growth. Thus, the functional loss of MsbA from the bacterium results in a toxic accumulation of lipid A within the inner membrane (3), making MsbA the only ABC transporter currently know to be required for bacterial viability (2).A reanalyzed crystal structure of the E. coli MsbA homodimer is now available at a resolution of 5.3 Å, along with the structures of MsbA from S. typhimurium and V. cholera and homologues such as SAV1866 (4-6). These structures reveal that the MsbA monomer contains † This work was supported by the National Institutes of Health (GM070642).*To whom correspondence should be addressed. Phone: (414) . E-mail: candice@mcw.edu. 1 Abbreviations: ABC, ATP-binding cassette; EPR, electron paramagnetic resonance; SDSL, site-directed spin labeling; CW, continuous wave; NBD, nucleotide binding domain; MTSL, 2,2,5,5-tetramethylpyrroline-3-yl-methanethiosulfonate spin label; WT,...

The Conformational Transition Pathway of ATP Binding Cassette Transporter MsbA Revealed by Atomistic Simulations

“…This step released the tetrahelix bundle from the constraints of the NBDs and facilitated the subsequent dissociation of the interaction network. It is worth noting that Glu-476 is a highly conserved residue on the x-loops among all ABC exporters (14), and cystine substitution of this residue greatly impairs the function of MsbA (53). Together with our simulation results, all of these highlight the structural and functional importance of x-loop.…”

“…The x-loop was recently identified as a conserved motif that precedes the LSGGQ motif only among the ABC exporters (14). Functional studies of both MsbA and TAP demonstrated that various mutations of the conserved glutamate in the x-loop reduced or completely abolished transport activity (53,54). Our MD simulation results showed that in the outward-facing conformation the conserved Glu-476 in the x-loop interacts with the cytoplasmic end of TM4 in cis through a salt-bridge of Glu-476-Lys-212, and this electrostatic interaction is important in pinning the NBD with the cytoplasmic tetrahelix bundle of TMD and stabilizing the tetrahelix bundle organization.…”

Time-resolved Fourier Transform Infrared Spectroscopy of the Nucleotide-binding Domain from the ATP-binding Cassette Transporter MsbA

“…The ATPase activity falls within the lower range of reported activities of the fulllength transporter (22,(51)(52)(53)(54). MsbA is found to be 2-4-fold stimulated by the binding of the allocrite lipid A to the transmembrane region, which is not included in our MsbA-NBD construct (19,51,53). A Hill coefficient of 1.5 indicates slight cooperativity in ATP binding and is consistent with values for the isolated NBD of the hemolysin transporter HlyB (43) and the mitochondrial transporter Mdl1p (44).…”

Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

“…In previous studies of full-length ABC protein exporters, functional and structural studies were performed under different experimental conditions. This is obvious in the case of crystallographic studies, but it is also true in the case of EPR spectroscopic studies, where function was evaluated at "normal" temperatures, but EPR data were obtained at very low temperatures (18,21,22). 2) To not only identify conformational states, but to perform kinetic studies of the transitions between those conformational states.…”