PT  - JOURNAL ARTICLE
AU  - Fangfang Jiang
AU  - Yasuharu Takagi
AU  - Arik Shams
AU  - Sarah M. Heissler
AU  - Thomas B. Friedman
AU  - James R. Sellers
AU  - Jonathan E. Bird
TI  - The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness
AID  - 10.1101/2020.06.17.155424
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.06.17.155424
4099  - http://biorxiv.org/content/early/2020/06/19/2020.06.17.155424.short
4100  - http://biorxiv.org/content/early/2020/06/19/2020.06.17.155424.full
AB  - Cochlear hair cells possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15A) traffics and delivers critical molecules required for stereocilia development and is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3. To understand the molecular mechanism of how MYO15A delivers proteins within stereocilia, we performed a kinetic study of the ATPase motor domain to characterize its mechano-chemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by co-expressing 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 co-expression had similar ATPase activities (kcat = ~ 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. This is consistent with MYO15 being adapted for strain sensing as a monomer, or processive motility if oligomerized into ensembles. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and ultimately disrupt stereocilia trafficking necessary for normal hearing.Competing Interest StatementThe authors have declared no competing interest.