Structural Biology of Formin Proteins

描述（由申请方提供）：形成蛋白是多结构域蛋白，参与所有真核生物中细胞极性、胞质分裂和形态发生所需的广泛细胞骨架过程。FH 2蛋白的定义特征是高度保守的400个残基区域，即形成蛋白同源2（FH 2）结构域，最近发现其直接使肌动蛋白丝成核。与Arp 2/3复合物不同，它使分支丝成核，formins诱导形成各种含肌动蛋白结构所需的无分支丝，包括收缩环，肌动蛋白电缆和应力纤维。“透明相关”formin（DRF）是作为Rho家族GTP酶的效应物的formin的子集。因为它们响应于不同的细胞信号而重组肌动蛋白细胞骨架，所以formins在细胞生物学和人类健康中具有核心重要性。β蛋白的缺陷导致胞质分裂失败和发育异常。人类CDFNA 1的缺陷会导致耳聋。我们的长期目标是在结构水平上了解肌动蛋白丝由α-肌动蛋白调节的组装。我们试图了解如何保守的cRFH 2结构域成核和锚定肌动蛋白丝，并了解内和分子间的相互作用，调节cRFH 2功能。我们正在使用X射线晶体学和其他生物物理方法来阐明酵母DRF Bni 1 p的结构和调节。如初步结果中所述，我们已经确定了Bnilp FH 2结构域的晶体结构。FH 2结构域的结构揭示了一种新的“拴系二聚体”结构，其中二聚体的两个半部分似乎是稳定的，但灵活地拴在一起。基于我们的初步结果，我们假设，不寻常的拴系二聚体结构的FH 2域允许formins通过稳定两个肌动蛋白亚基在肌动蛋白丝的倒刺端发现的螺旋方向成核肌动蛋白丝，并在新生丝的倒刺端，因为它的生长阶梯步骤。在这里，我们建议通过在体外和体内的结构/功能研究，以测试所产生的假设的结构，并确定与肌动蛋白复合物的FH 2结构域的结构，以详细阐明肌动蛋白组装的机制，由formin。此外，我们将通过Bni 1 p内自抑制区域的晶体学分析来扩展这项工作，以了解它们如何调节FH 2结构域。

DESCRIPTION (provided by applicant): Formins are multidomain proteins that participate in a wide range of cytoskeletal processes that are required for cell polarity, cytokinesis, and morphogenesis in all eukaryotes. The defining feature of formin proteins is a highly conserved - 400 residue region, the Formin Homology 2 (FH2) domain, which has recently been found to directly nucleate actin filaments. Unlike the Arp2/3 complex, which nucleates branched filaments, formins induce unbranched filaments required for formation of diverse actin-containing structures including the contractile ring, actin cables, and stress fibers. The "Diaphanous-related" formins (DRFs) are a subset of formins that are effectors for Rho-family GTPases. Because they reorganize the actin cytoskeleton in response to diverse cellular signals, formins are of central importance in cell biology and to human health. Defects in formin proteins result in failed cytokinesis and abnormal development. Defects in the human formin DFNA1 result in deafness. Our long-term goal is to understand at a structural level the regulated assembly of actin filaments by formin proteins. We seek to understand how the conserved formin FH2 domain nucleates and anchors actin filaments and to understand the intra- and inter- molecular interactions that regulate formin function. We are using X-ray crystallography and other biophysical methods to elucidate the structure and regulation of the yeast DRF Bni1 p. As described in the preliminary results, we have determined the crystal structure of the Bnilp FH2 domain. The structure of the FH2 domain reveals a novel "tethered-dimer" architecture, in which the two halves of the dimer appear to be stably, but flexibly tied together. Based on our preliminary results, we hypothesize that the unusual tethered-dimer construction of the FH2 domain allows formins to nucleate actin filaments by stabilizing two actin subunits in the helical orientation found at the barbed end of actin filaments, and to stair-step on the barbed end of the nascent filament as it grows. Here we propose to test hypotheses generated by the structure through in vitro and in vivo structure/function studies, and to determine the structure of the FH2 domain in complex with actin in order to elucidate in detail the mechanism of actin assembly by formins. Furthermore, we will extend this work with crystallographic analysis of autoinhibitory regions within Bni1p in order to understand how they regulate the FH2 domain.