MECHANISM OF ACTIN FILAMENT NUCLEATION BY VIBRIO PARAHEMOLYTICUS VOPL

副溶血弧菌 VOPL 肌动蛋白丝成核机制

• 批准号: 8361288
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 0.59万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361288
• 关键词: Actins; Binding; Biochemical; Biophysics; C-terminal; Cell Nucleus; Complex; Databases; Dimerization; Distal; Funding; Grant; Microfilaments; Minus End of the Actin Filament; Names; National Center for Research Resources; Principal Investigator; Proteins; Research; Research Infrastructure; Resources; Roentgen Rays; Solutions; Source; Structure; United States National Institutes of Health; Vibrio parahaemolyticus; beamline; cost; dimer; insight; novel; polymerization

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We performed Small Angle X-ray Scattering (SAXS) structural studies using the BioCAT beamline on a novel actin filament nucleator from Vibrio parahaemolyticus named VopL. We have found that the strong actin filament nucleation activity of VopL depends on the presence of three WH2 domains and dimerization through a C-terminal domain (DD) of unknown structure. Since this DD had no detectable similarity to any known structure in the Protein Data Bank, we used SAXS to study its structure in solution to gain further insights into the nucleation mechanism of VopL. The SAXS structure showed the expected two-fold symmetry of the dimer, consisting of a central dimerization 'knot domain', which probably comprises the predicted C-terminal coiled coil, and two diametrically opposed smaller domains. We also studied the complex of actin with VopL fragment 1W-DD (containing one of the three WH2 domains and the DD).. While the study of this complex did not yield much new information, a few inferences may be drawn: a) the structure of the DD reemerged as a stable unit in the structure of the complex with actin, b) actin subunits are bound loosely in the polymerization nucleus and c) the actin subunits bind at the interface between the central knob and small distal domains. In summary, our biochemical and structural analyses by SAXS of VopL constructs ¿actin suggest that the dimerization domain binds at the pointed end of the actin filament and contains a coiled-coil region at the C-terminus.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们使用BioCAT光束线对副溶血性弧菌一种名为VopL的新型肌动蛋白细丝核仁进行了小角X射线散射(SAXS)结构研究。我们发现，VopL强大的肌动蛋白细丝成核活性依赖于三个WH2结构域的存在和通过未知结构的C-末端结构域(DD)的二聚化。由于该DD与蛋白质数据库中的任何已知结构都没有可检测到的相似性，因此我们使用SAXS研究了它在溶液中的结构，以进一步了解VopL的成核机制。SAXS结构显示了预期的二聚体的两重对称性，包括一个中心二聚化‘结结结构域’，它可能包括预测的C-末端卷曲线圈，以及两个截然相反的较小结构域。我们还研究了肌动蛋白与VopL片段1W-DD(包含三个WH2结构域之一和DD)的络合物。虽然对该复合体的研究没有产生太多新的信息，但可以得出一些推断：a)DD的结构在含有肌动蛋白的复合体的结构中重新出现为一个稳定的单位，b)肌动蛋白亚单位松散地结合在聚合核中，c)肌动蛋白亚单位结合在中央旋钮和远端小结构域之间的界面上。综上所述，我们对VopL构建的肌动蛋白进行的SAXS生化和结构分析表明，二聚化结构域结合在肌动蛋白细丝的尖端，并在C末端包含一个卷曲的区域。