Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration, proliferation and survival. In cancer FAK is a major driver of invasion and metastasis and its upregulation is associated with poor patient prognosis. FAK is autoinhibited in the cytosol, but activated upon localisation into a protein complex, known as focal adhesion complex. This complex forms upon cell adhesion to the extracellular matrix (ECM) at the cytoplasmic side of the plasma membrane at sites of ECM attachment. FAK is anchored to the complex via multiple sites, including direct interactions with specific membrane lipids and connector proteins that attach focal adhesions to the actin cytoskeleton. In migrating cells, the contraction of actomyosin stress fibres attached to the focal adhesion complex apply a force to the complex, which is likely transmitted to the FAK protein, causing stretching of the FAK molecule. In this review we discuss the current knowledge of the FAK structure and how specific structural features are involved in the regulation of FAK signalling. We focus on two major regulatory mechanisms known to contribute to FAK activation, namely interactions with membrane lipids and stretching forces applied to FAK, and discuss how they might induce structural changes that facilitate FAK activation.

Significance In the dynamic environment of the airways, where SARS-CoV-2 infections are initiated by binding to human host receptor ACE2, mechanical stability of the viral attachment is a crucial fitness advantage. Using single-molecule force spectroscopy techniques, we mimic the effect of coughing and sneezing, thereby testing the force stability of SARS-CoV-2 RBD:ACE2 interaction under physiological conditions. Our results reveal a higher force stability of SARS-CoV-2 binding to ACE2 compared to SARS-CoV-1, causing a possible fitness advantage. Our assay is sensitive to blocking agents preventing RBD:ACE2 bond formation. It will thus provide a powerful approach to investigate the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.