The SecA translocation ATPase is the engine that powers the ATP-driven extrusion of secreted proteins from the eubacterial cytoplasm. It has been hypothesized that SecA acts like a molecular ratchet in this process, effecting transmembrane transport of substrate polypeptides by pulling them into and through the phospholipid bilayer concomitant with its own ATP-driven membrane insertion/retraction cycle. The goal of the proposed research is to clarify how this remarkable molecular machine works in terms of the structural mechanics of the membrane insertion/transport reaction and the thermodynamic coupling of these protein conformational changes to the ATPase cycle of the enzyme. The recently completed crystal structure of the soluble form of SecA (J.F. Hunt, S. Weinkauf, L. Henry, D.B. Oliver, and J. Deisenhofer, manuscript in preparation) provides a starting point for these studies and has allowed specific structural hypotheses to be formulated for some features of the chemo-mechanical cycle of the enzyme. This structure shows extensive and intimate packing interactions between the four domains in the SecA monomer, and, interpreted in the context of established biochemical results, suggests that a nucleotide-modulated domain-dissociation reaction controls the transition from the soluble, compact conformation of SecA observed in the crystal to a more extended conformation where individual domains of SecA are free to penetrate into the phospholipid bilayer and/or interact with downstream effectors in the translocation pathway. Fluorescence anisotropy and resonance energy transfer measurements will be used to characterize the relative movement of the SecA domains during the ATPase cycle and the membrane-inserted state: Spectroscopic studies will be conducted with intact SecA and with isolated transmembrane domains in order to characterize their secondary structure and disposition relative to the phospholipid bilayer in the membrane-embedded state, crystallographic studies will be attempted with the isolated cytoplasmic domains complexed with ligands that stabilize them in the membrane-associated conformation. This approach should enable us to reconstruct a comprehensive structural and thermodynamic picture of the chemo-mechanical transport cycle of SecA. Beyond the long-term potential for this information to be put to use in designing biomolecular machines with novel activities, another by-product of the proposed research will be to develop a simple and efficient assay for high-throughput screening of anti-SecA inhibitors; because SecA is the known physiological target of the broad-spectrum bacteriostat azide (N3-) and because a homologous enzyme does not occur in animal cells, efficacious inhibitors of SecA are likely to represent promising lead compounds for the development of novel anti-bacterial drugs.
