Investigation of the interactions between collagen-binding integrins and collagen : binding and structural studies of a 1l domain and collagen mimetic peptides

Integrins comprise of a large family of heterodimeric cell adhesion receptors consisting of an α- and a β- subunit. They are responsible for mediating cell-cell and cell-extracellular matrix (ECM) interactions. In addition, they transmit bi-directional (outside-in and inside- out) signals across the plasma membrane, regulating numerous important cellular behaviours including cell adhesion, cell migration and cell communication. Collagen-binding integrins represent one of the five integrin subfamilies and are characterized by their ligand specificity for different collagens. Each α subunit in the collagen-binding subfamily contains an inserted domain, the αI domain, which is responsible for collagen binding. The interactions between two of the most studied collagen-binding integrins, α1β1 and α2β1, and their respective collagen substrates have been implicated in several disease processes, making these receptors attractive therapeutic targets. The initial aim of this project was to identify small molecule scaffolds that are suitable starting points for designing inhibitors of the α1β1-collagen interaction. The approach adopted was fragment-based drug discovery. The lack of structural information on the collagen-bound state of α1I domain, however, made the process very challenging. Therefore, it was decided that the structure of the α1I domain-collagen complex should be characterised to support any subsequent program of rational drug design from the fragment-based approach. A variety of techniques were employed to characterise the interactions between α1I domain and synthetic collagen peptides. These included surface plasmon resonance spectroscopy (SPR) which provided details of the affinity and kinetics of binding, size exclusion chromatography and small angle X-ray scattering that revealed an unexpected stoichiometry of binding, NMR experiments that enabled the structure of α1I domain to be determined in the presence of a collagen peptide and computer docking using the data-driven docking program, HADDOCK, to generate a model of the entire α1I domain-collagen peptide complex. Combining the information generated from all these techniques in conjunction with previously reported structural and mutagenesis studies, a detailed insight into the interaction was provided. Not only do these results provide a structural rationale to design inhibitors of collagen binding to α1I domain, they are also valuable for understanding the biological roles and the regulation of this receptor.