Scaffold Protein Tethering Ability under Load: FAK's FERM Domain Mechanical Properties V. Binding Site

Abstract

Mechanotransductive scaffolding proteins are tethered and thus subjected to mechanical loads that potentially induce partial or total unfolding. Focal adhesion kinase (FAK) is regulated by mechanical stimulation through extracellular matrix (ECM) proteins and actin cytoskeleton contractility. FAK is composed of three major domains: two of which putatively perform tethering (the FERM and FAT domains) while the central kinase domain is catalytically active in a wide variety of cell motility/invasion pathways upon activation. The so-called “basic patch” is the ligand-binding site on FERM's F2 subdomain, which is connected, via an unstructured loop, directly to FERM's F3 subdomain and distally to the kinase. As a mechanically competent tether, the FERM domain must carry loads between the basic patch and the F3 subdomain's C-terminal. A key question is whether these subdomains lose their tertiary structure under load_and therefore unwrap into a “beads on a string” configuration_and, if so, what consequences this has for ligand-binding subdomain stability. Towards an understanding of the FERM domain's ability to tether a mechanically competent FAK (pdb: 2al6), FERM's unfolding pathways are studied via Steered Molecular Dynamics (SMD). SMD simulations of the unfolding process reveal force peaks, extended conformations of intermediate states, and intra-molecular load pathways. Loads are applied to FERM's C-terminal and a set of residues in the basic patch known to bind to both phospholipids and phosphopeptides. By differentially applying loads to the basic patch's secondary structures, unfolding behavior, including both force levels and intermediate states, is revealed. Pulling-mode simulations_mimicking AFM_identify unfolding intermediate states; constant-force-mode simulations probe the structural behavior of identified intermediates. Given the diversity of ligands known to bind to the basic patch, mechanical behavior as a function of binding-site secondary structure is crucial for understanding mechanotransduction.