The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness.
The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness.
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DOI：
10.1074/jbc.ra120.014903
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Bird JE
中科院分区：
文献类型：
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作者：
Jiang F;Takagi Y;Shams A;Heissler SM;Friedman TB;Sellers JR;Bird JE
每一个耳蜗毛细胞都拥有一束精致的肌动蛋白静纤毛，可以探测声音。非常规肌球蛋白15（MYO 15）运输和提供静纤毛发育所需的关键分子，因此对于建立机械感觉毛束至关重要。人类MYO 15 A基因的突变会干扰静纤毛运输并导致遗传性听力损失（DFNB 3），但这些突变的影响尚不清楚，因为MYO 15本身的特征很差。为了了解更多，我们进行了ATP酶运动域的动力学研究，以表征其机械化学循环。使用杆状病毒-Sf 9系统，我们通过共表达小鼠MYO 15 ATP酶、结合其IQ结构域的必需和调节轻链以及ATP酶正确折叠所需的UNC 45和HSP 90 A分子伴侣来纯化重组最小马达结构域（S1）。用UNC 45 A或UNC 45 B共表达纯化的MYO 15具有相似的ATP酶活性（20 °C时kcat = 1.66 s-1）。使用停流和猝灭流瞬态动力学分析，我们测量了描述ATP酶循环的主要速率常数，包括ATP，ADP和肌动蛋白结合;水解;和磷酸盐释放。肌动蛋白连接的ADP释放是最慢的测量转换（在20 °C下为1012 s-1），尽管这并没有限制ATP酶循环的速率。动力学分析表明MYO 15马达结构域具有中等的占空比（0.5），ADP与肌动蛋白结合的热力学耦合较弱。这些发现与MYO 15在寡聚化时在动力学上适应进行性运动一致。我们的动力学表征使未来的研究能够了解致聋突变如何影响MYO 15并破坏听力所需的静纤毛运输。
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–Sf9 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 (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.