Landscape-Based View on the Stepping Movement of Myosin VI

[Image: see text] Myosin VI dimer walks toward the minus end of the actin filament with a large and variable step size of 25–36 nm. Two competing models have been put forward to explain this large step size. The Spudich model assumes that the myosin VI dimer associates at a distal tail near the cargo-binding domain, which makes two full-length single α-helix (SAH) domains serve as long legs. In contrast, the Houdusse–Sweeney model assumes that the association occurs in the middle (between residues 913 and 940) of the SAH domain and that the three-helix bundles unfold to ensure the large step size. Their consistency with the observation of stepping motion with a large and variable step size has not been examined in detail. To compare the two proposed models of myosin VI, we computationally characterized the free energy landscape experienced by the leading head during the stepping movement along the actin filament using the elastic network model of two heads and an implicit model of the SAH domains. Our results showed that the Spudich model is more consistent with the 25–36 nm step size than the Houdusse–Sweeney model. The unfolding of the three-helix bundles gives rise to the free energy bias toward a shorter distance between two heads. Besides, the stiffness of the SAH domain is a key factor for giving strong energetic bias toward the longer distance of stepping. Free energy analysis of the stepping motion complements the visual inspection of static structures and enables a deeper understanding of underlying mechanisms of molecular motors.