Mechanical forces and ligand-binding modulatePseudomonas aeruginosaPilY1 mechanosensitive protein

Abstract

Bacteria initiate colonization and biofilm formation in response to mechanical cues caused by surface proximity. The protein PilY1 has been proposed as a key actor mediating mechanosensing. PilY1 is a calcium and integrin-binding protein with additional roles in host adhesion and functional regulation of the type IV pili (T4P), the appendages involved in twitching motility, and various aspects of the surface-associated life of bacteria. Due to its extracellular location and involvement in several surface processes, PilY1 is exposed to mechanical forces that could modulate its different roles. Herein, we explore the effect of mechanical forces and ligand binding on the conformational dynamics of the PilY1 C-terminal domain. Our single-molecule approach demonstrates that PilY1 acts as a ligand-modulated force sensor. At high forces, PilY1 unfolding occurs through a hierarchical sequence of intermediates. When calcium is bound to its cognate site linked to T4P regulation, there is a long-range mechanical stabilization affecting several PilY1 domains, which ensures the structural integrity of the protein. In the low-force regime, the integrin-binding domain of PilY1 exhibits calcium-tuned force sensitivity and conformational dynamics akin to those of mechanosensor proteins. Integrin binding to this domain occurs under force, inducing a shortening of its unfolded extension. Our findings suggest that the roles of the PilY1 C-terminal domain are force and ligand-modulated, which could entail a mechanical-based compartmentalization of its functions.