Structural and mechanistic dissection of the myosin start-of-power stroke state and its importance for force production

• Applicants: Professor Dr. Matthias Preller; Professor Dr. Georgios Tsiavaliaris
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 322867023

• Final Report Year: 2021

Final Report Abstract

Over the past years our groups have been extensively working on functional and structural aspects of myosins. Helpful in this respect has been the continuous improvement and progress in protein production, the implementation and further development of a broad spectrum of kinetic and functional studies in combination with computer-assisted protein engineering, molecular modelling and X-ray structural analyses. The applied methods and techniques have formed a solid basis for studying the actomyosin system from various aspects including the cellular functions of the motors, and the development of lead compounds targeting myosin involved in diseases. In the funded project, the collaboration of our groups has resolved molecular details of mechanotransduction yet not or only partially resolved by previous approaches. The key achievements of the funded project include: 1. Deciphering force generating communication pathways in the myosin motor a. Linking the actin-binding region via the W-helix with the active site; b. Connecting the active site with the mechanical converter. 2. Experimental evidence for the start-of-power stroke state as an essential intermediate for force production. 3. Contribution of the regulatory light chains in the allosteric coupling mechanism of actomyosin communication. 4. Elucidation of the high-resolution structure of an ADP-release conformation of myosin related to the two-step mechanism of force generation. 5. Mechanisms and regulation of actin dynamics and muscle performance resolved by molecular dynamics simulations of the tropomyosin-actin complex. 6. Development and application of a new generation of thermophoresis-based phosphate and photoacoustic calcium biosensors for studying enzyme catalysis and mechanisms of cell regulation. The funded project has significantly strengthened the interaction of our groups and has highly benefitted from our individual and complementary expertise. A major achievement has been to uncover details of the complex mechanism of actomyosin communication following a comprehensive and multidisciplinary approach, made possible by the synergy of our labs. Our results allow to further refine the myosin ATPase cycle, initially proposed by Lymn and Taylor, and to quantitatively describe the events that dictate the propagation of mechanochemical coupling signals, which eventually generate force.

Publications

• (2017). Diatrack particle tracking software: review of applications and performance evaluation. Traffic, 18(12), 840-852
  Vallotton, P., van Oijen, A.M., Whitchurch, C.B., Gelfand, V., Yeo, L., Tsiavaliaris, G., Heinrich, S., Dultz, E., Weis, K., and Grünwald, D.
  
• (2017). Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C. Elife, 6, e32742
  Chinthalapudi, K., Heissler, S., Preller, M., Sellers, J., Manstein, D.J.
  
• (2017). MicroScale Thermophoresis (MST) for studying actin polymerization kinetics. BioTechniques. 63 (4), 187-190
  Topf., A., Franz, P., and Tsiavaliaris, G.
  
• (2018) Calcium sensor for photoacoustic imaging. J. Am. Chem. Soc., 140, 2718-2721
  Roberts S., Seeger M., Jiang Y, Mishra A., Sigmund F., Stelzl A, Lauri A., Symvoulidis P., Rolbieski H., Preller M., Deán-Ben X.L., Razansky D., Orschmann T., Desbordes S.C., Vetschera P., Bach T., Nziachristos V., Westmeyer G.G.
  
• (2019). Profilin2a-phosphorylation as a regulatory mechanism for actin dynamics. FASEB J.
  Walter, L.S., Franz, P. Lindner, R., Tsiavaliaris, G., Hensel, N., and Claus, P.
  
• (2019). Transformation of the Nonprocessive Fast Skeletal Myosin II into a Processive Motor. Small, 15
  Amrute-Nayak, M., Nayak, A., Steffen, W., Tsiavaliaris, G., Scholz, T., and Brenner, B.
  
• (2020) A role for actin flexibility in thin filament-mediated contractile regulation and myopathy. Nat. Commun., 11, 2417-2432
  Viswanathan, M.C., Schmidt, W., Franz, P., Rynkewiecz, M.J., Newhard, C.S., Madan, A., Lehman, W., Swank, D.M., Preller, M. and Cammarato, A.
  
• (2020) A thermophoresis-based biosensor for real-time detection of inorganic phosphate during enzymatic reactions. Biosens. Bioelectron. 169:112616
  Franz P., Gassl V., Topf A., Eckelmann L., Iorga B., and Tsiavaliaris G.
  
• (2020) Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin-II function. J. Biol. Chem.
  Nayak A., Wang T., Franz P., Steffen W., Chizhov I., Tsiavaliaris G., Amrute-Nayak M.
  
• (2020) Structural and computational insights into a blebbistatin-bound myosin·ADP complex with characteristics of an ADP-release conformation along the two-step myosin power stoke. Int. J. Mol. Sci.; 21(19):7417
  Ewert W., Franz P., Tsiavaliaris G., and Preller M.
  
• (2020). Small molecule effectors of myosin function. In Advances in Experimental Medicine and Biology - Myosins, Springer Nature Ltd, ISBN: 978-3-030-38061-8
  Manstein, D.J. and Preller, M.
  
• (2021) Unraveling a force-generating allosteric pathway of actomyosin communication associated with ADP and Pi release. Int. J. Mol. Sci., 22(1): 104
  Franz P., Ewert W., Preller M., Tsiavaliaris G.