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

Cochlear hair cells each 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 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 MYO15A itself is poorly characterized. To learn more, 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. 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. Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Citation

Fangfang Jiang, Yasuharu Takagi, Arik Shams, Sarah M Heissler, Thomas B Friedman, James R Sellers, Jonathan E Bird. The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness. The Journal of biological chemistry. 2021 Jan 08:100243

PMID: 33428946

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