The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Abstract

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus– Sf 9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities ( k cat = ∼ 6 s −1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s −1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing

“…Second, EGFPtagged MYO15-2 actively traffics elongation complex proteins along filopodia in cell lines (Belyantseva et al, 2005;Manor et al, 2011;Mauriac et al, 2017). Finally, enzymatic studies of the purified MYO15 ATPase domain reveal kinetic adaptations that enable long-range processive molecular trafficking (Bird et al, 2014;Jiang et al, 2021). Together, these data support MYO15-2 delivering the elongation complex to the stereocilia tips, where the elongation complex is hypothesized to regulate actin polymerization.…”

“…To study the interaction between MYO15 and actin filaments in more detail, we characterized the influence of the jordan mutation upon ATPase mechanochemistry (Bird et al, 2014;Jiang et al, 2021). Motor domain proteins were expressed in S. frugiperda (Sf9) insect cells and purified by chromatography (Fig.…”

Propagation of F-actin disassembly via Myosin15-Mical interactions

“…These results are consistent with work in other systems where proteins such as dyneins and kinesins drive long-range movements along microtubules, whereas myosins mediate short-range movements along actin filaments ( 22 ). Previous work using the purified motor domain of class XV myosins reveals that Myo15 is a high–duty ratio molecular motor [i.e., the myosin head (motor) spends its time strongly bound to its F-actin track during each ATPase cycle, a property required for processive movement of myosins on F-actin] and that the Myo15 motor moves rapidly (400 nm/s) with a relatively short 8-nm power stroke toward the membrane-proximal barbed end of F-actin ( 30 , 31 ). Therefore, these previous observations, coupled with our results in Figs.…”

Functional Role of Class III Myosins in Hair Cells

“…However, the long MYO15A isoform is able to traffic to these bundles and prevent disassembly, suggesting a role in stereocilia architecture and maintenance. Recent kinetic analysis of the MYO15A motor demonstrated a moderate duty ratio (∼0.5) and relatively high affinity for actin (Jiang et al, 2021). In vitro motility experiments demonstrated that MYO15A can move actin filaments at rate of ∼300-400 nm/s and confocal microscopy showed localization of MYO15A at protrusion tips of live cells (Belyantseva et al, 2005;Bird et al, 2014).…”

“…There are two possible models for MYO15A that can explain its ability to act as a transporter within stereocilia. One such model suggests that MYO15A functions as a dimer or oligomer, perhaps as a complex with cargo, to move processively toward the actin protrusion tips (Jiang et al, 2021). The second model suggests a membrane binding mediated movement, whereby monomeric MYO15A associates with both the actin cytoskeleton and the plasma membrane, moving via a diffusive sliding mechanism (Bird et al, 2014).…”