Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VIIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VIIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca(2+) but not high Ca(2+). By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low Ca(2+) suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head.

There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. K(ATPase) of the actin-activated ATPase was 62 microm, which is much higher than that of myosin Va ( approximately 1 mum). The rate of ADP release from actomyosin Vc was 12.7 s(-1), which was 2 times greater than the entire ATPase cycle rate, 6.5 s(-1). P(i) burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms.