Localization and functional exploration of leiomodin-2's C-terminal binding sites

Striated muscle contraction occurs through interactions between overlapping myosin-based thick and actin-based thin filaments within the sarcomere. For effective contraction to occur, the length of the thin filament must be maintained to ensure sufficient overlap with the thick filament. The proteins leiomodin and tropomodulin compete for binding at the pointed ends of thin filaments to regulate their length, using their homologous N-terminal actin and tropomyosin binding sites. Leiomodin also contains a region called the C-terminal extension, absent in tropomodulin. In this region, the cardiac isoform (leiomodin-2) contains additional actin-binding sites that enable it to bind along the sides of thin filaments in a Ca 2+ -dependent manner. Here, using nuclear magnetic resonance spectroscopy, we localize the regions of the C-terminal extension that contain residues involved in thin filament side-binding. Using co-sedimentation assays, we reveal that these regions can bind thin filaments independently of one another and discover that the poly-proline region serves as a linker, maintaining an adequate distance between two of the regions required for effective interaction. In addition to its role in side-binding, we provide evidence that the poly-proline region interacts with profilin and propose a new mechanism by which leiomodin-2 may assist in the polymerization of profilin-bound actin at thin filament pointed ends. • Lmod2's poly-proline region binds profilin-1. • Lmod2's poly-proline region is required for effective thin filament side-binding. • Lmod2's thin filament side-binding sites within residues 374–426, 451–506, 531–550. • Lmod2's thin filament side-binding sites can bind independently of one another.