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

Myosin motor function plays a pivotal role in processes of cellular motility and muscle contraction resulting from the cyclic interaction of the myosin with actin filaments. Mechanical work and directed movement is generated during the myosin power stroke - a rotational movement of the long lever-arm - which is fuelled by the hydrolysis of ATP. This process constitutes the key event of myosin motor activity. However, our knowledge about the molecular events of mechanotransduction remains incomplete. The lack of structural information of conformational states that occur prior to, during, and subsequent to the power stroke, as well as an insufficient description of the allosteric communication pathways within the motor domain has yet prevented a full description of the mechanotransduction mechanism in molecular detail. With the aid of a set of selected mutations, which we will introduce within functional regions of the motor domain, the current research program aims at refining and complementing the mechanochemical cycle of the acto-myosin interaction according to Lymn and Taylor. We identified these mutations using computational simulations to selectively perturb the allosteric coupling between actin binding and force generation via the W-helix/transducer region and consequently trap the motor in conformations representative for the start-of-power stroke, i.e. a yet structurally unresolved high actin-affinity state with the actin binding cleft closed but different from the classical rigor state with the lever-arm in an up position. Among the specified mutations producing the desired effects are mutations that are known to cause cardiomyopathies. We will characterize these mutations on the protein level using a broad spectrum of state-of-the-art biophysical and biochemical methods and complement the analyses with mechanistic simulations and X-ray crystallography. As a result we expect to obtain high-resolution structures of those elementary states in the cycle that could so far not been structurally resolved with native or unmodified proteins and gain fundamental insights into the mechanism of how actin contributes to the power stroke and drives product release in molecular detail. The major outcome of the synergy between our laboratories will be to map the impact of new communication pathway by relating the changes in the kinetic and thermodynamic features caused by the mutations to structural aspects, which in turn will allow us to quantitatively describe the events that dictate the propagation of mechanochemical coupling signals and eventually generate force. The knowledge about the exact communication pathways is not only essential for understanding the molecular mechanisms of myosin dysfunction in diseases, including cardiomyopathies and neurosensory disorders but it also will promote the development of highly specific therapeutic drugs that act in an allosteric manner to inhibit or activate myosin performance in those diseases.

• DFG Programme: Research Grants