SAXS STUDY ON THE CYTOPLASMIC SUBCOMPLEX FROM THE VIBRIO CHOLERAE TYPE 2 SECRETI

2型霍乱弧菌细胞质亚复合体的SAXS研究

• 批准号: 7721820
• 负责人: Jan Abendroth
• 金额: $ 0.11万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721820
• 关键词: ATP phosphohydrolase; Binding; Cholera Toxin; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Cytoplasmic Tail; Electrons; Funding; Grant; Infectious Agent; Institution; Link; Measures; Membrane; Membrane Proteins; Nucleotides; Organism; Pattern; Pilum; Positioning Attribute; Reporting; Research; Research Personnel; Resources; Rest; Screening procedure; Solutions; Source; System; Thinking; Type II Secretion System Pathway; United States National Institutes of Health; Vibrio cholerae; Vibrio parahaemolyticus; Vibrio vulnificus; dimer; pathogenic bacteria; size

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 霍乱弧菌等病原菌使用II型分泌系统通过外膜输出其感染性物质，如霍乱毒素。当孔位于外膜时，该系统由细胞质分泌ATPase(EPSE)提供能量。EPSE通过双功能内膜蛋白EPSL与系统的其余部分相连。来自相关系统的各种报道，如III型分泌物、IV型菌毛，表明核苷酸结合对EPSE的寡聚态有显著影响，并且寡聚态对ATPase活性有影响。然而，有迹象表明，EPSE与EPSL胞浆结构域的复合体(Cyto-EPSL)的寡聚模式与目前的学说不同。我们有证据表明，在核苷酸存在的情况下，EPSE(E)和Cyto-EPSL(CL)至少形成E2cL2，很可能形成E6cL6的寡聚体。我们已经开始进行溶液X射线散射研究，以研究这些组装作为不同核苷酸的函数，并补充我们正在进行的关于这个体系的结晶学研究。我们对E-CL复合体进行了两次测量，得到的大小略有不同：Rg~43A为500 kDa，Rg~38A为400 kDa。电子对分布函数在相同的原子间距离处有峰值位置，这表明两种形式的络合物都有共同的组装单元，很可能是E-Cl二聚体。我们研究了ATP对来自不同生物的E-CL复合体的影响，因为它们被认为在结晶筛选中表现得更好。加入三磷酸腺苷后，副溶血性弧菌(VPO)复合体的寡聚状态没有改变：RG44.4vs43.8A(+ATP)。创伤弧菌(VV)的另一个复合体也表现出类似的微小变化：RG38.8vs39.8A(+ATP)。虽然到目前为止，我们还没有观察到任何依赖于ATP的低聚物组装的实质性变化，但这些结果表明，有两种不同的低聚物组装形式，一种是Rg~43-44A，另一种是38-39A。