Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily due to 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 which primarily associates with calmodulin, we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, calmodulin, and calmodulin like protein 4 (CALML4). Our results further reveal that CALML4 does not function as a Ca 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 which have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechano-enzymatic 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 such as MyRIP. 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.
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