Structure of the Leiomodin-2 Regulated Actin Filament Pointed End Assembly from Profilactin

Cardiac contraction depends on synchronized interactions between myosin-based thick filaments and actin-based thin filaments (TFs). Precise regulation of TFs length is vital for cardiac function, as any alteration in length leads to severe myopathies. Actin filaments form the backbone of the TF and have two unequal ends - fast-growing barbed and slow-growing pointed. In muscle, the barbed end is capped at the Z-line, while the pointed end is regulated by the tropomodulin family of proteins. Tropomodulin caps the pointed end, while leiomodin-2 (Lmod2) promotes actin nucleation and pointed end elongation. Lmod2 has a unique C-terminal extension (CTE) that is important for actin nucleation and binds to the sides of matured TFs. The structural mechanism by which Lmod2 promotes elongation remains elusive. We employed cryo-electron microscopy to visualize the structure of growing pointed ends nucleated by Lmod2 from profilactin. We show that Lmod2's leucine-rich repeat domain (LRR) stabilizes terminal actin subunits by binding across the helical groove of actin. We identified two distinct populations of pointed-end LRR-containing complexes on one or both actin strands. LRR binding pushes the terminal actins outward from their ideal positions in the actin filament, introducing strain at the pointed end that squeezes LRR from the filament's exterior. We also show that the Lmod2 CTE may stabilize Lmod2 binding to the pointed end. We suggest that Lmod2 promotes the addition of new actins to the pointed end but is expelled from the growing filament, thereby maintaining the concentration of Lmod2 required for further elongation.