Structural and functional characterization of the myosin VI tail

Myosin VI is the only known molecular motor that moves towards the minus end of actin filaments, and thus plays pivotal roles in diverse cellular processes, including endocytosis, Golgi network maintenance and excytosis, epithelia cells polarity establishment and so on. Myosin VI contains the motor domain in the N-terminus, following by two IQ domains responsible for light chain binding, and the C-terminal class-specific tail domain (including the commonly assumed coiled-coil domain and the cargo binding domain, in short CBD). Processive walking of myosin VI on actin filaments requires the motor to dimerize. At the same time myosin VI is also known to function as nonprocessive monomer in cells. It is believed that the tail domain plays critical roles in the monomer-dimer conversion, while the detail mechanisms remain largely unclear. It is also known that myosin VI motor has a large step size up to 36 nm in spite of its very short lever arm. It is still unresolved how myosin VI can walk with such large steps with its unusually short legs. Here, in my PhD study, I systematically explore the structure and function on the tail domain of myosin VI. I discovered that the CBD undergoes a cargo (Dab2)-binding mediated dimerization. The cargo-mediated dimmerization likely represents a new paradigm in the regulation of processivities for myosin VI as well as other unconventional myosins, including myosin VII and myosin X. The N-terminus of the tail domain (referred to as the lever arm extension, LAE), which is previously termed as “coiled-coil domain”, adopts a stable monomeric, three-helix bundle fold in solution. More importantly, the myosin VI LAE undergoes reversible, lipid membrane-dependent conformational changes. Upon exposure to lipid membranes, the myosin LAE adopts a partially extended rod shape composed of three α-helices jointed together by two restrained and retrievable connectors. The removal of lipid from the LAE converts the domain back into a compact three-helix bundle fold. This reversible, lipid membrane-dependent expansion of the LAE, together with the previously identified “IQ” motifs and the extended single helix rod following the LAE, provides enough space and flexibility for myosin VI to walk on actin filaments with large and variable step sizes. In summary, the work described in this thesis solved two major questions for myosin VI that have been challenging scientists for the past decades