This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Pathogenic bacteria such as Vibrio cholerae employ Type II Secretion Systems for the export of their infectious agents such as cholera toxin through the outer membrane. While the pore is located in the outer membrane, the system is energized by a cytoplasmic secretion ATPase (EpsE). EpsE is linked to the rest of the system via the bitopic inner membrane protein EpsL. Various reports from related systems, e.g. Type III Secretion, Type IV Pili, indicate that nucleotide binding has a dramatic effect on the oligomeric state of EpsE, and that the oligomeric state has an effect on the ATPase activity. However, there are indications that the oligomerization pattern of EpsE in complex with the cytoplasmic domain of EpsL (cyto-EpsL) differs from the current dogma. We have evidence that EpsE (E) and cyto-EpsL (cL) form oligomeric assemblies at minimum E2cL2, and likely E6cL6, in the presence of nucleotides. We have begun conducting solution x-ray scattering studies to investigate these assemblies as a function of different nucleotides and complement our on-going crystallographic studies on this system. We have measured E-cL complex in two occasions, and obtained slightly different sizes: 500kDa with Rg~43A and 400kDa with Rg~38A. The electron pair distribution functions have the peak position at the identical inter-atomic distance, suggesting both forms of complex have common assembly unit, likely E-cL dimer. We studied the effects of ATP on E-cL complex from different organisms as they are thought to behave better in crystallization screening. Upon addition of ATP, the complex from Vibrio parahaemolyticus (Vpo) complex did not change its oligomerization state: Rg 44.4 vs 43.8A (+ATP). The other complex from vibrio vulnificus (VV) showed similarly minute change: Rg 38.8 vs 39.8A (+ATP). Although we have not so far observed any substantial ATP-dependent change in oligomer assembly, these results indicate that there are two distinctive forms of oligomer assembly, one with Rg~43-44A and the other with 38-39A.
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