The objective is to elucidate steps in ATP driven cycles that lead to pumping substrates across membranes of the cell using high-resolution cryo-electron microscopy. Atomic structures determined by X-ray crystallography, serial femtosecond X-ray, spectroscopic rulers, and antibodies are used as tools to define an integrative model of this dynamic process that will provide for new ways of countering drug resistance or disease conditions.
The objective is to understand the basis for energy driven cycles that lead to pumping substrates against the gradient of their concentration, across membranes of the cell. The Program Project approach is to determine the structures of the distinct functional states in the multi-step transport cycles and the pathways between them without relying exclusively on crystallography, since crystallizing intermediate states of very different structure is often as difficult as crystallizing the initial structure. The Program stratgy combines crystallography, that provides an atomic resolution structures, with specific Fab fragments to aid in sub nanometer electron cryo- microscopy (cryo-EM), `temperature dependent' cryo-EM, super-resolution Fluorescence Energy Transfer (FRET) spectroscopy, double electron-electron Resonance (DEER), serial femtosecond x-ray diffraction (SFX), small angle X-ray scattering (SAXS), chemical and disulfide cross-linking, and integrative structure modeling methods. The project focuses on ABC transporters that use ATP binding at two sites and ATP hydrolysis as the energy source for transport of substrates. The Program aims are to define the mechanism of coupling ATP binding to transport in single transporters. To accomplish this the structures of a heteromeric exporter, a homodimeric peptide exporter, and a heteromeric multi-drug exporter are expressed and will be subject to structure determination. Each transporter will be stalled at certain states throughout the transport cycle with some 5-6 expected states verified by pumping assays, or trapped by femtosecond X-ray pulses synchronized to light flash activation. Antibody Fab fragments will be generated by screening libraries displayed in bacteriophage against stabilized states of the cycle. The Fab fragments provide additional orientation for high- resolution cryo-EM imaging that provides domain interactions, Fab locations, detergent and lipid locations. X- ray crystallography provides the atomic basis for interpreting the domains, which are placed accurately within cryo-EM images. Mutations are introduced to provide for distance-sensitive labels and spectroscopies that define distances between selected points through critical stages in the mechanism. These data are subject to integrative structure modeling that seeks to then produce the pathway between the states, revealing the currently undefined mechanism of ABC transporters at atomic level. In humans 48 ABC transporters coordinate normal physiology. Through understanding the structural basis for moving through many states new target conformations for human therapeutics will be uncovered. This integrative approach and simulations of the pumping cycle consistent with thermodynamics of the cycle will be applicable to many other large complexes of membrane proteins.