Structural insights into F-actin regulation and sarcomere assembly via myotilin

Kostan, J.; Pavsic, M.; Puz, V.; Schwarz, T. C.; Drepper, F.; Molt, S.; Graewert, M. A.; Schreiner, C.; Sajko, S.; van der Ven, P. F. M.; Svergun, D.; Warscheid, B.; Konrat, R.; Fuerst, D. O.; Lenarcic, B.; Djinovic-Carugo, K.

doi:10.1101/2020.09.25.310029

bioRxiv · 10.1101/2020.09.25.310029

Structural insights into F-actin regulation and sarcomere assembly via myotilin

Kostan, J.·Pavsic, M.·Puz, V.·Schwarz, T. C.·Drepper, F.·Molt, S.·Graewert, M. A.·Schreiner, C.·Sajko, S.·van der Ven, P. F. M.·Svergun, D.·Warscheid, B.·Konrat, R.·Fuerst, D. O.·Lenarcic, B.·Djinovic-Carugo, K.

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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 -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

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Kostan, J., Pavsic, M., Puz, V., Schwarz, T. C., Drepper, F., Molt, S., Graewert, M. A., Schreiner, C., Sajko, S., van der Ven, P. F. M., Svergun, D., Warscheid, B., Konrat, R., Fuerst, D. O., Lenarcic, B., Djinovic-Carugo, K.. 2020-09-25. Structural insights into F-actin regulation and sarcomere assembly via myotilin. https://doi.org/10.1101/2020.09.25.310029

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