Focal adhesion kinase (FAK) controls the assembly of focal adhesion sites and transduces signals from several membrane receptors. Controlled activation and localization of FAK functionally links cell adhesion, migration and survival. FAK is overexpressed in many cancer types, promoting tumor invasiveness and metastasis. The molecular mechanisms allowing FAK to fulfil numerous different functions and act as versatile ‘nanomachines’ are poorly understood. We have previously revealed that ligand-induced dimerization along with intramolecular interactions control FAK activation and localization where the C-terminal focal adhesion targeting (FAT) domain is strictly involved. In this study, we combine NMR with X-ray crystallography, as well as biophysical and computational methods to understand the molecular mechanisms that link the large-scale dynamics and intramolecular and intermolecular interactions of FAT into FAK’s capacity to integrate various stimuli into a site-specific function. Our results reveal FAT-mediated dynamical interplays between binding of known and newly discovered FAT ligands, and multimerization and autoactivation of FAK. Additionally, we investigate the impact of neuronal alternative splicing on FAT dynamics and interactions. Collectively, our results elucidate FAT’s role in allosterically controlling various FAK functions, and might inspire allosteric protein-protein interaction inhibitors against FAK-dependent cancer cell proliferation.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase. Activated FAK is crucial to many biological processes, such as cell proliferation, migration, and survival, all of which have been implicated in the progression and development of cancer. Tyrosine 925 is a Src-phosphorylation site that is located within the FAT domain in the C-terminal of FAK. It has been suggested that the helix containing Y925 (Helix 1) has to come out of the FAT bundle and the region flanking Y925 has to adopt β-strand conformation. In order to phosphorylate, the mechanisms promoting the required structural changes are unclear. So, Molecular Dynamics (MD) and Constant Force Molecular Dynamics (CFMD) simulations were used to study what makes Y925 accessible for phosphorylation. Under thermal fluctuation only and in the presence or the absence of LD motifs, MD simulations suggest that H1 does not appear to have a propensity to leave the bundle adopt β-strand conformation. Then, two different load scenarios were used; axial and perpendicular with 100 pN constant load applied to H1 N-terminus with the two paxillin LD motifs constrained. For both load scenarios, H1 has two different behaviors: typical and atypical. In the axial load scenario, the first two residues at the N-terminal of H1 (besides Y925) have low propensity to unfold. However, H1 does not show any proclivity to leave the bundle. For the perpendicular load scenario with the absence of P2 (LD motif binds to H1/H4 hydrophobic patch), one simulation out of 21 showed that H1 undergoes the required structural rearrangement. In general, CFMD simulations show that the FAT domain has a very low propensity (3%) to undergo the structural changes needed for Y925 phosphorylation. This has two implications: either mechanical load is insufficient to make Y925 available for phosphorylation and/or this kind of process (structural changes needed for Y925 phosphorylation) is slow process that needs a long time to occur. / text