Mechanical forces and ligand binding modulate PilY1 mechanosensitive protein.

Surface sensing initiates bacterial colonization of substrates. The protein PilY1 plays key roles during this process-surface detection, host adhesion, and motility-while experiencing mechanical perturbations of varying magnitudes. In , the adhesion and motility functions of PilY1 are associated with integrin and calcium ligand-binding sites; however, how mechanical forces influence PilY1's dynamics and its interactions with these ligands remain unknown. Here, using single-molecule magnetic tweezers, we reveal that PilY1 is a mechanosensor protein that exhibits different behaviors depending on the force load. At high forces (>20 pN), PilY1 unfolds through a hierarchical sequence of intermediates, whose mechanical stability increases with calcium binding. This enhanced stability may help counteract type IV pilus retraction forces during motility. At low forces (<7 pN), we identify the dynamics of the integrin-binding domain, which is reminiscent of the behavior of mechanosensor proteins. Integrin binding induces a force-dependent conformational change in this domain, shortening its unfolded extension. Our findings suggest that PilY1 roles are force- and ligand-modulated, which could entail a mechanical-based compartmentalization of its functions.