Structural studies on the talin head domain - a key regulator of cell-matrix interactions
Lead Research Organisation:
University of Liverpool
Department Name: Sch of Biological Sciences
Abstract
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.
Technical Summary
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.
Publications
Goult BT
(2013)
Structural studies on full-length talin1 reveal a compact auto-inhibited dimer: implications for talin activation.
in Journal of structural biology
Goult BT
(2010)
Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation.
in The EMBO journal
Goult BT
(2009)
The structure of the N-terminus of kindlin-1: a domain important for alphaiibbeta3 integrin activation.
in Journal of molecular biology
Goult BT
(2013)
RIAM and vinculin binding to talin are mutually exclusive and regulate adhesion assembly and turnover.
in The Journal of biological chemistry
Goult BT
(2010)
The domain structure of talin: residues 1815-1973 form a five-helix bundle containing a cryptic vinculin-binding site.
in FEBS letters
Gingras A
(2010)
Central Region of Talin Has a Unique Fold That Binds Vinculin and Actin
in Journal of Biological Chemistry
Elliott PR
(2010)
The Structure of the talin head reveals a novel extended conformation of the FERM domain.
in Structure (London, England : 1993)
Bouaouina M
(2012)
A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated aIIbß3 integrin coactivation.
in The Journal of biological chemistry
Description | Talin is a key adhesion molecule that is essential for the activation of integrin receptors. We discovered that the structure of the talin head is unique in the family of the related FERM domain proteins. Four domains of the talin head make a linear extended arrangement, in contrast to a previously observed compact structure in other proteins. This linear arrangement is optimal for the binding to the cell membrane and for integrin activation. The linear arrangement is stabilised by the linker between the two central domains in the talin head and we proposed a set of mutations that can modulate the inter-domain arrangement and thus affect integrin activation. We identified strcutural features of the talin head that independently contribute into the integrin activation. (i) All the domains include positively charged residues that are aligned on on side of the linear structure and make the membrane interaction dependent on the negative surface charge. (ii) The second domain of the head includes a large insert loop that dynamically adopts a helical structure in the presence of negatively charged membrane. Combining our finding into a model of integrin activation we propose a "fly-casting" mechanism where the insert loop initiates the membrane contact and subsequently pulls the rest of the talin head close to the membrane surface. On the membrane the positively charged surface of the molecule is engaged, aligning the talin head in the optimal position for the integrin activation. Our work provides molecular details for the integrin activation by talin and identifies the critical components of the process that can be manipulated to modulate cell adhesion and migration. |
Exploitation Route | The results are immediately relevant to the fundamental understanding of the cell-matrix adhesion. In the future, the interactions of the talin head could be exploited to control cell adhesion and cell migration. |
Sectors | Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
Description | Results were used in the undergraduate and postgradual teaching. NMR analysis approaches developed in the study were applied in translational projects. |
First Year Of Impact | 2010 |
Sector | Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Title | Expression vectors for talin head fragments |
Description | DNA plasmids for expressing recombinant talin fragments in e.coli for in vitro analysis. Detains published in "The structure of the talin head reveals a novel extended conformation of the FERM domain. Paul R. Elliott, Benjamin T. Goult, Petra M. Kopp, Neil Bate, J. G?nter Grossmann , Gordon C. Roberts, David R. Critchley and Igor L. Barsukov (2010) Structure, 18, 1289-1299" "Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation." Benjamin T. Goult, Mohamed Bouaouina, Paul R. Elliott, Neil Bate, Bipin Patel, Alexandre R. Gingras, J. G?nter Grossmann, Gordon C.K. Roberts, David A. Calderwood, David R. Critchley and Igor L. Barsukov (2010) EMBO J, 29, 1069-1080 |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2010 |
Provided To Others? | Yes |
Impact | facilitated experiments |
Title | Crystal structure of the talin head FERM domain, 3IVF |
Description | PDB database deposition of the atomic coordinates of the strcuture of the whole talin head |
Type Of Material | Database/Collection of data |
Year Produced | 2009 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.rcsb.org |
Title | NMR structure of the F0 domain (residues 0-85) of the talin ferm domain |
Description | PDB database deposition of the atomic coordinates of the structure of the talin head F0 domain |
Type Of Material | Database/Collection of data |
Year Produced | 2008 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.rcsb.org |
Title | NMR structure of the F0F1 double domain (residues 1-202) of the talin ferm domain |
Description | PDB database deposition of the atomic coordinates of the structure of the talin head F0F1 region |
Type Of Material | Database/Collection of data |
Year Produced | 2009 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.rcsb.org |
Title | Solution structure of the talin F3 in complex with layilin cytodomain |
Description | PDB database deposition of the atomic coordinates of the structure of the talin head F3 domain complex |
Type Of Material | Database/Collection of data |
Year Produced | 2008 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.rcsb.org |
Description | NMR analysis of kindlin |
Organisation | Yale University |
Country | United States |
Sector | Academic/University |
PI Contribution | The collaboration is a follow up from talin studies. Critical for the understanding of the role of kindlin in adhesion. |
Start Year | 2010 |
Description | Role of talin in force transduction |
Organisation | Yale University |
Department | School of Medicine |
Country | United States |
Sector | Academic/University |
PI Contribution | Use of a combination of structural biology and fluorescence microscopy to define the effect of force on the talin interactions in adhesion. |
Start Year | 2010 |
Description | Structural and functional studies of Shank3 |
Organisation | University Medical Center Hamburg-Eppendorf |
Department | Institute for Human Genetics |
Country | Germany |
Sector | Hospitals |
PI Contribution | Identified new functional domain in autism-related proteins Shank3 and solved its structure using information and approached of the funded project. |
Collaborator Contribution | Partners demonstrated the role of Shank3 in the integrin regulation through collaborative research. |
Impact | Structure of Shank3 SPN domain, PDB ID 5G4X. Publication DOI 10.1038/ncb3487 |
Start Year | 2013 |
Description | Structural and functional studies of Shank3 |
Organisation | University of Turku |
Department | Turku Centre for Biotechnology |
Country | Finland |
Sector | Academic/University |
PI Contribution | Identified new functional domain in autism-related proteins Shank3 and solved its structure using information and approached of the funded project. |
Collaborator Contribution | Partners demonstrated the role of Shank3 in the integrin regulation through collaborative research. |
Impact | Structure of Shank3 SPN domain, PDB ID 5G4X. Publication DOI 10.1038/ncb3487 |
Start Year | 2013 |
Description | Structural and functional studies of talin |
Organisation | University of Manchester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | The collaboration links in vitro analysis of talin with cell studies. Critical for the understanding of the role of talin in adhesion. Generated further publications and grant applications in the adhesion area. Stimulates multi-disciplinary research. |
Collaborator Contribution | Partners conducted experiments in cells to test predictions from structural analysis in vitro. |
Impact | Multi-disciplinary collaboration, partners conduct research in cell biology field. Publications, DOI: 10.1038/ncomms10038, 10.1016/j.str.2016.04.016, 10.1074/jbc.M112.438119 |
Start Year | 2011 |
Description | Talin link to the integrin regulation |
Organisation | University of Turku |
Country | Finland |
Sector | Academic/University |
PI Contribution | Structural analysis on the novel regulator of integrin activity. |
Start Year | 2010 |
Description | Gordon Research Conference 2011 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Participants in your research and patient groups |
Results and Impact | Presentation at the research conference no actual impacts realised to date |
Year(s) Of Engagement Activity | 2011 |