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

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.

High-resolution structure of the talin head domain (residues 1-400) will be determined by a combination of NMR and SAXS and the effect of solvent conditions such as pH and salt concentrations on the structure will be analysed. The contributions from individual subdomains of the talin head into the interactions with layilin and integrin PIP kinase will be characterised by NMR mapping of the interaction sites and comparison of the affinities of different talin fragments for the ligands measured by stopped-flow fluorescence, ITC and NMR. Talin binding sites for the complete layilin cytodomain will be defined by the NMR mapping and the structure of the full talin-layilin complex will be determined. The correlation between the binding of different talin head ligands will be established. PIP2 binding sites on the talin head subdomains will be identified and the structures of the PIP2 complexes with the individual subdomains and complete talin head with be determined. The effect of the PIP2 binding on the conformation of the talin head and its interaction with ligands will be established. Conditions for the talin complex assembly in a membrane-like environment will be optimised and an experimental model that reconstructs multi-component talin complexes will be assembled. The results of the project will be integrated to design talin mutations that selectively affect interaction with ligands either through direct interaction or indirectly, through modulation of subdomain arrangement.