Structural studies on the talin head domain - a key regulator of cell-matrix interactions

在所有多细胞生物体中，细胞都附着在一种特殊的组织--细胞外基质上。这建立了生物体的完整性，并塑造了其他组织，使细胞-基质相互作用对胚胎发育和组织维持至关重要。细胞通过嵌入在细胞膜外层的整合素受体与基质相连。在连接受体和细胞骨架的受体的胞内区形成了一个大型的多蛋白黏附复合体，维持了细胞的形状和刚性。在移动细胞时，通过一系列相连的事件与基质建立联系。在前缘，细胞与LAYLIN受体建立最初的联系。在细胞内，一种接头蛋白talin被招募到粘连的位置。Talin反过来又结合并激活PIP激酶，后者在粘连部位产生信号分子PIP2。此外，Talin还能激活紧密接触所需的整合素受体。Talin还在受体和肌动蛋白细胞骨架之间建立了联系，使细胞能够施加运动所需的力。研究这样一个复杂的系统时，主要的挑战是获得一个综合的信息，其中考虑了所有的组成部分。为了实现这一点，我们选择了黏附复合体的关键蛋白质-talin，并设计了一系列实验，使用各种生物物理方法来分析talin在黏附的不同阶段形成的相互作用网络。我们的目标是提取指导和调节Talin相互作用的主要因素，对它们进行深入分析，然后将这些知识整合到一个可以进行实验测试的单一模型中。我们集中在这个长达2541个残基的蛋白质的头部区域，因为它对与整合素和Laylin受体的相互作用至关重要，并且包含PIP激酶和PIP2的结合部位。这个400个残基的片段由四个定义明确的亚域组成，我们的初步数据表明，亚域的相对取向可能取决于蛋白质环境，这为活性调节提供了一种机制。首先，我们将确定全塔林头的结构，并分析影响它的因素。然后我们将引入Talin配体，并确定Talin头部不同部分对相互作用的贡献。我们预计一些亚域将参与直接接触，而另一些亚域将通过影响结合域而间接做出贡献。我们还将研究一个配体的结合对Talin与不同配体相互作用的影响。这些信息对于理解在黏附复合体组装过程中一个talin配体如何取代另一个talin配体是至关重要的。尽管Talin与PIP2的黏附调节很重要，但由于复合体的低稳定性，Talin与PIP2的相互作用仍然难以捉摸。我们在前期研究中找到了分析PIP2复合体的条件，并将确定PIP2结合对talin结构的影响，以了解PIP2激活talin的机制。由于黏附复合体组装在膜上，在确定膜的作用之前，对该体系的理解是不完整的。我们将得到一些条件，使我们能够在膜状环境中获得结构信息，并利用这些条件重建塔林黏附复合体。这将把所有主要成分结合在一起，并将为Talin功能的综合分析提供一个实验模型。我们将使用该模型来设计talin突变，以选择性地增强特定的相互作用，以便它们可以与细胞实验中的生物学特性相关联。

In all multi-cellular organisms cells are attached to a special tissue, extracellular matrix. This establishes the integrity of the organism and shapes other tissues, making cell-matrix interaction essential for the embryonic development and tissue maintenance. The cells are attached to the matrix through integrin receptors that are embedded in the outer cell membrane. A large multi-protein adhesion complexes are formed at the intra-cellular domains of the receptors connecting receptors to the cytoskeleton that maintains the shape and rigidity of cells. In moving a cell the contacts with the matrix are established through a series of connected events. At the leading edge the cell engages layilin receptors to establish initial contacts. Inside the cell an adaptor protein talin is recruited to the site of the adhesion. Talin, in turn, binds and activates PIP kinase that generates signalling molecule PIP2 at the site of the adhesion. In addition, talin activates integrin receptors that are required for a strong contact. Talin also makes a connection the between the receptors and actin cytoskeleton that allows the cell to apply force required for the motion. The main challenge when studying such a complex system is to derive a comprehensive information that takes all components into account. In order to achieve this we have chosen a key protein of the adhesion complex - talin and devised a set of experiments using various biophysical methods to analyse the network of interactions that talin forms at different stages of adhesion. We aim at extracting the main factors that direct and regulate talin interaction to analyse them in depth and then to integrate this knowledge within a single model that can be tested experimentally. We concentrate on the head domain of this large 2541-residue protein as it is crucial for the interaction with integrin and layilin receptors and contains binding sites for PIP kinase and PIP2. This 400-residue fragment consists of four well-defined subdomains and our preliminary data indicate that the relative orientation of the subdomains may depend on the protein environment, providing a mechanism for the activity regulation. Initially we will determine the structure of the full talin head and analyse the factors that affect it. We will then introduce talin ligands and determine the contribution from different parts of the talin head into the interactions. We expect that some of the subdomains will be involved in direct contact, while others will contribute indirectly by affecting the binding domains. We will also study the effect of binding of one ligand on the talin interaction with a different ligand. This information is essential for the understanding of how one talin ligand displaces another during the adhesion complex assembly. Despite of the importance for the adhesion regulation, talin interaction with PIP2 remains elusive due to the low stability of the complex. We found conditions for the PIP2 complex analysis in our pilot studies and will determine the effect of PIP2 binding on talin structure in order to understand the mechanism of talin activation by PIP2. As the adhesion complexes are assembled on a membrane, the understanding of the system is incomplete until the effect of the membrane is determined. We will derive conditions that will allow us to obtain structural information in a membrane-like environment and will use them to reconstitute the talin adhesion complexes. This will bring all the main components together and will provide an experimental model for the integrated analysis of the talin function. We will use the model to design talin mutations that selectively enhance specific interactions so they can be correlated with the biological properties in cell experiments.