DESCRIPTION (provided by applicant): The class of proteins identified as ATP-binding cassette (ABC) transporters is one of the largest found in nature. Their ability, or lack thereof, to move a variety of ligands across a membrane bilayer using energy from ATP is fundamentally important to bacterial physiology and to an array of human pathologies. ABC transporters mediate both the import and export of a wide variety of solutes including antibiotics, chemotherapy agents, and lipids. Specifically, MsbA is the ABC transporter for lipid A that is found in the inner membranes of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, and Vibrio cholerae. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A, which is an essential component of the outer surface of the cells, within their inner membranes. Although crystal structures have been solved for MsbA, the original structures were retracted and the reanalyzed data were recently republished as new structures. Still, many questions remain concerning its functional structure. And, the exact mechanisms of function on the molecular level during the catalytic cycle remain largely unidentified in ABC transporters in general, and the mechanism by which MsbA transports lipid A has yet to be established. The local dynamics of regions key to substrate binding have been determined in MsbA by our group in the last funding period, adding detailed knowledge of this essential protein. The role of each site responsible for the function or dysfunction of ATP hydrolysis and transport of lipid A is valuable information not readily attainable by other methods at this time. Identifying the specific steps in which many of these conserved sites play in the transport process will greatly improve our current functional understanding of this important class of transporters. Insights into the function of both fully active as well as dysfunctional transporters are key to our fundamental understanding of an entire class of potential drug and gene therapy targets. Thus, the proposal that the MsbA homodimer undergoes significant local and global conformational rearrangements through changes in the location of the dimer interface and within the nucleotide binding domains upon ATP and lipid A binding that are essential to its function as a lipid A exporter will be tested using a step by step approach to function using a combination of biochemical activity assays and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy techniques. PUBLIC HEALTH RELEVANCE: Multidrug resistance is a serious problem in medicine, not only in the often futile treatment of infectious diseases, but also in the treatment of cancer patients. In addition, because of the close similarity of MsbA with other ABC transporters implicated in various common genetic disorders such as the cystic fibrosis transmembrane conductance regulator, any new functional information gained by studying the easily purified MsbA will be beneficial to our understanding of structure-function relationships in this very important class of integral membrane proteins. The similarity of MsbA to so many proteins in its general class, and its prevalence in pathogenic bacteria, creates an opportunity for the detailed study of this transporter that will add to our fundamental knowledge of an entire class of potential novel drug targets.

Project Narrative Multidrug resistance is a serious problem in medicine, not only in the often futile treatment of infectious diseases, but also in the treatment of cancer patients. In addition, because of the close similarity of MsbA with other ABC transporters implicated in various common genetic disorders such as the cystic fibrosis transmembrane conductance regulator, any new functional information gained by studying the easily purified MsbA will be beneficial to our understanding of structure-function relationships in this very important class of integral membrane proteins. The similarity of MsbA to so many proteins in its general class, and its prevalence in pathogenic bacteria, creates an opportunity for the detailed study of this transporter that will add to our fundamental knowledge of an entire class of potential novel drug targets.