Molecular basis of F-actin regulation and sarcomere assembly via myotilin

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain containing protein myotilin provides structural integrity to Z-discs - the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and -actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. We used a combination of small angle X-ray scattering, cross-linking mass spectrometry, biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilins structural role in Z-discs. Significance StatementSarcomeres are the primary structural and functional unit of striated muscles, conferring movement in all animals. The Z-disk is the boundary between adjacent sarcomeres, where actin filaments (F-actin) are anchored. Z-disc protein myotilin, is a scaffold protein, which provides structural integrity to the Z-disc by multiple interactions to its central components, including F-actin and -actinin-2. Here we provide the structure of myotilin, revealing its structural plasticity in solution and the first integrative structural model of its complex with F-actin. We further show that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond being an interaction hub for Z-disk partners. Highlights Myotilin is structurally described as a dynamic ensemble Flanking regions enhance F-acting binding to tandem Ig domains Integrative structural model of myotilin bound to F-actin Myotilin displaces tropomyosin from F-actin, suggesting an organisational role in Z-disc