Molecular regulatory mechanism of human myosin-7a.

Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm's structural-functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.

The kinetic mechanism of mouse myosin VIIA.

Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s(-1) is activated by actin only by ∼3.5-fold resulting in a V(max) of 0.7 s(-1) and a K(ATPase) of 11.5 µM. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T M·D·P) is slow (12 s(-1)) and the equilibrium constant (K(H)) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s(-1). Phosphate dissociation is relatively fast (>12 s(-1)), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 µM because of the weak binding of the myosin VIIa-ADP-P(i) complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s(-1)) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a "slow", monomeric molecular motor with a duty ratio of 0.6.