Molecular basis of F-actin regulation and sarcomere assemblyviamyotilin

Abstract

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 α-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 myotilin’s 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 α-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.HighlightsMyotilin is structurally described as a dynamic ensembleFlanking regions enhance F-acting binding to tandem Ig domainsIntegrative structural model of myotilin bound to F-actinMyotilin displaces tropomyosin from F-actin, suggesting an organisational role in Z-disc

Myofilament-associated proteins with intrinsic disorder (MAPIDs) and their resolution by computational modeling

“…Another excellent MAPID study that utilized both experiment and simulation was performed by Kostan et al . (2021). Conformations comprising myotilin's ensemble were selected from a pool of structures generated by the ‘EOM’ approach (Bernadó et al ., 2007) to match experimental SAXS profiles.…”

“…Conformations comprising myotilin's ensemble were selected from a pool of structures generated by the ‘EOM’ approach (Bernadó et al ., 2007) to match experimental SAXS profiles. These conformations, together with biochemical binding assays, provide an integrative structural model that rationalizes the mechanism, by which myotilin's IDR binds to F-actin (Kostan et al ., 2021).…”

“…Michie et al (2016) used a similar strategy to identify a linker in the M-domain of MyBPC3 for binding calmodulin via SAXS scattering and computational modeling. Another excellent MAPID study that utilized both experiment and simulation was performed by Kostan et al (2021). Conformations comprising myotilin's ensemble were selected from a pool of structures generated by the 'EOM' approach (Bernadó et al, 2007) to match experimental SAXS profiles.…”