Focal adhesion kinase (FAK) controls adhesion-dependent cell motility survival and proliferation. motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment which occurs specifically at focal adhesions. Paxillin plays a dual role by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. protein-protein interface rather than a crystal packing artifact (solvation free energy gain [Δof 29?μM (95% confidence interval 17 for the FERM:FERM interaction. We then produced full-length WT FAK as a His6 fusion protein in a baculovirus system purified it on a Ni2+ column (Supplementary Fig S2C) and examined its ability to self-associate. Polyacrylamide gel electrophoresis under native conditions revealed a dimeric species of full-length FAK which disappeared in the presence of increasing amounts of SDS (Fig?(Fig4A)4A) or in W266A-FAK (Fig?(Fig4B).4B). Dynamic light scattering (DLS) experiments indicated the presence of two major FAK species with Stokes radii (through FERM:FERM conversation including W266 Representative native PAGE of His6-FAK Clomipramine HCl (loaded concentration 4?μM) followed by immunoblot with an anti-FAK monoclonal antibody showing monomeric (*) and dimeric (°) … SAXS analysis of full-length FAK confirms FERM:FERM interactions and suggests Excess fat:FERM interactions The size flexibility and low solubility of FAK have so far precluded structural analysis of the full-length molecule by X-ray crystallography nuclear magnetic resonance or electron microscopy. We therefore used SAXS to investigate the intramolecular interactions occurring in full-length FAK in answer. We first used an iterative approach to determine low-resolution CD209 bead models. The SAXS data were compatible with the particle size expected for any FAK dimer (Fig?(Fig5A5A Clomipramine HCl and ?andB B Supplementary Fig S3) and the averaged structure displayed dimeric features even without symmetry constraints (Supplementary Fig S3A). The central part of the SAXS bead model was highly similar to the arch shape of the crystallographic FERM:FERM dimer. Moreover the crystallographic FERM-kinase homodimers (PDB 2J0K or 2J0J) fitted well into a large part of the SAXS bead model (Fig?(Fig5A).5A). After this fit the SAXS envelope provided additional space near the FERM F2 lobe likely to harbor many of the ∽400 residues missing in the FERM-kinase structure. In a second SAXS analysis we used the homodimeric FERM-kinase fragment as a rigid body and placed the FAT domain and the other missing Clomipramine HCl residues (the 38?N-terminal residues and the 220 kinase-FAT linker residues) directly according to the SAXS scattering pattern. This algorithm did not take into account the SAXS bead model. In the 10% best-scored of the ∽500 produced models the four-helical FAT domain was consistently placed in contact with the FERM F2 lobe close to the site where FERM binds to the C-terminal kinase lobe suggesting a direct conversation between the FERM and FAT domains (Fig?(Fig5A 5 Clomipramine HCl Supplementary Fig S3C). The DAMMIN bead model (gray spheres) is shown superimposed onto the molecular … FAT binds to a basic cluster on FERM and thus stabilizes the FAK dimer Since the FAT:FERM interaction suggested by our SAXS study had not been reported previously Clomipramine HCl we tested it of 0.6?±?0.2?μM (Supplementary Fig S4A). We also observed the association between immobilized FAT and GST-FERM or FAT and immobilized GST-FERM by SPR (Supplementary Fig S4B). The putative Excess fat binding region recognized by SAXS within the FERM F2 lobe encompassed Clomipramine HCl a basic cluster K216AKTLRK which is usually important for FAK function (Dunty and requires W266 Having shown that FAK can form dimers through FERM:FERM and Excess fat:FERM contacts we examined the functional relevance of this self-association. Autophosphorylation of Y397 is the central step in activation of kinase-dependent functions of FAK. Previous studies suggested that in intact cells and and requires dimerization through W266 and in cells Specific autophosphorylation activity (i.e. the ratio of pY397 to FAK immunoblot signals).