Interactions and Functions of Myosin 16 Domains

The unconventional myosin 16b (Myo16b), which may have a role in the neuronal development and the regulation of cell cycle, can be found both in the nucleus and in the cytoplasm. Myo16b contains a motor domain, a short neck region, an itrinsically disordered tail (My16Tail) and an ankyrin-repeat containing N-terminal domain, called the ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g. in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c) and My16Ank shows a sequence similarity to MYPT1. We characterized the Myo16b domains: the My16Ank, the motor domain, and the My16Tail in vitro using recombinant protein fragments. We tested the effects of My16Ank on the myosin motor function using skeletal muscle myosin as a model system. The results showed that My16Ank bound to skeletal muscle myosin (KD ∼ 2.4 µM) and the actin-activated ATPase activity of the skeletal myosin was increased in the presence of My16Ank. Besides, we showed, that My16Ank strongly bound to PP1cα (KD ∼ 540 nM) and also to PP1cδ (KD ∼ 600 nM) isoforms. The prolin-rich My16Tail is supposed to bind profilin, but we found no direct interaction between My16Tail and fluorescent profilin-1 using anisotropy measurements. Meanwhile, we also tested the interaction between My16Tail and My16Ank domains, which showed a moderate affinity (KD ∼ 13 µM). Our results suggest that one function My16Ank is probably to regulate the motor function of Myo16. It may influence the motor activity and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, yet unidentified, intracellular proteins. My16Tail can bind My16Ank assuming a regulatory function by backfolding of the tail to the N-terminal of the Myo16.