| Abstract |
The cell constitutively synthesizes proteins. 30% of the proteome is extracytoplasmic rendering secretion as a basic process of the cell life. In Bacteria, the experimental system that we use in this thesis, exist a variety of secretory machines (translocases). Transocases with the expenditure of energy, either in the form of ATP or in the form of gradient of protons in the membrane, secretes proteins through the lipid bilayer towards the extracellular space. From all the above secretory machines, only Sec translocase is essential for life, since it secretes the majority of the exracytoplasmic proteins and that is why it is studied in more detail in this analysis. The basic core of Sec translocase consists of the SecYEG oligomeric pore-like structures in the membrane and the peripheral protein SecA. Although SecA is a processive enzyme moving along preprotein secreted substrates, it shares homology with nucleic acid helicases. More specifically it contains a helicase amino terminal catalytic core (N-domain) with two nonhomologous specificity domains protruding from it. While the N-domain performs almost all the chemistries of SecA (ATP binding and hydrolysis, binding of the Signal Petide of the substrate and binding to the membrane at SecYEG), the so called specificity domains help helicases acquire their specific role and in particular assign SecA its task in protein secretion. These are: the Substrate Specificity Domain, SSD, where the preprotein substrate is postulated to bind and the Carboxy terminal C-domain that we have found it regulates the ATPase activity of the N-catalytic domain. The C-domain, through direct binding to the N-catalytic domain, suppresses SecA ATPase when it stays in the cytoplasm and theres no need for energy consumption. Respectively in the absence of a tight association between the N-/C-domains, SecA ATPase is activated without any binding from ligands, mimicking the SecA behaviour in the membrane during translocation, when ATP hydrolysis is needed for secretion to happen. The binding of the C-domain to the N-domain is attributed to the first C-domain substructure of the Scaffold Domain, SD, but we have found also that IRA1 (Intramolecular Regulator of ATP hydrolysis 1) a conserved Helix-Loup-Helix structure in the C-domain is required for its regulatory role. SecA with a short IRA1 deletion becomes a hyperactivated ATPase that is nevertheless incompetent for transocation. Trying to answer the unresolved question of energy conversion to mechanical work in helicases, that most probably involves cross-talk between the catalytic and specificity domains, we focused on IRA1 mechanism, that is expected to act as a molecular switch essential for coupling ATP hydrolysis to translocation work. A variety of biochemical data supported also by the recently solved structures of SecA from Bacillus.subtilis and Mycobacterium.tuberculosis, suggest that through direct or long range conformational communication with almost all SecA subdomains, IRA1 senses all the ligands of the translocation reaction. Consequently IRA1 transmits conformational changes to the N-catalytic domain, regulating its properties. We propose that IRA1 intramolecular regulation is exerted through lateral oscillations of IRA1 to and from the helix of the SD that binds directly to the catalytic domain. Through these oscillations IRA1 affects SDs conformation and controlls its binding and release from the N-domain. Likewise intermolecular regulation of SecA by the preprotein substrates is expected to involve similar -oscillations- of IRA1 to and from the SSD since IRA1 is positioned between the SD and the SSD, and it's the only physical link connecting them. Analogous H-L-H substructures in helicases in general, could potentially connect their specificity domains and couple catalysis to mechanical work.
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