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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们使用 BioCAT 光束线对来自副溶血弧菌的一种名为 VopL 的新型肌动蛋白丝成核剂进行了小角 X 射线散射 (SAXS) 结构研究。我们发现 VopL 的强肌动蛋白丝成核活性取决于三个 WH2 结构域的存在以及通过未知结构的 C 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没有可检测到的相似性，因此我们使用 SAXS 研究其在溶液中的结构，以进一步了解 VopL 的成核机制。 SAXS 结构显示出预期的二聚体双重对称性，由中心二聚化“结域”（可能包含预测的 C 端卷曲线圈）和两个完全相反的较小域组成。我们还研究了肌动蛋白与VopL片段1W-DD的复合物（包含三个WH2结构域之一和DD）。虽然对该复合物的研究没有产生太多新信息，但可以得出一些推论：a）DD的结构在与肌动蛋白的复合物结构中重新出现为稳定单元，b）肌动蛋白亚基松散地结合在聚合核中，c）肌动蛋白亚基在聚合核处结合 中央旋钮和小远端区域之间的界面。总之，我们通过 SAXS 对 VopL 构建体肌动蛋白进行的生化和结构分析表明，二聚化结构域结合在肌动蛋白丝的尖端，并在 C 末端包含一个卷曲螺旋区域。