Expression Studies of Other Unconventional Myosins

National Heart, Lung, And Blood Institute

Investigators

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Abstract

We have begun to analyze the structure and stepping pattern of myosin-6, a processive myosin which moves in the opposite direction on actin compared to other myosins. EM studies show that the angle between the two heads is more variable than in most myosins and that when bound to actin, the motors can be spaced at 13 actins (preferred) or 11 or 15 actin monomers apart. Optical trapping and EM studies demonstrate that the molecule can sometimes take an "inchworm" like step where the two heads occupy closely spaced binding sites. 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 governs 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 Ca2+ sensor but plays a crucial role in maintaining the lever arms 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 the sensory systems such 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. In collaboration with John Hammer we have begun to study myosin 19 a mitochondrial associated myosin to complement ongoing cell biological studies.