Mammalian myosin-18 is encoded by two genes, MYO18A and MYO18B. The MYO18A gene products are reported as two splice variants, and , that are essential to various developmental and tumor suppression activities. They have been reported to have roles in Golgi localization, maintenance of cytoskeleton, and receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif and a long coiled-coil reminiscent of myosin-2. Myosin-18A comprises an additional N-terminal extension containing a PDZ domain. To conduct a biochemical analysis of M. musculus myosin-18, recombinant HMM-, and S1-like 18A and 18A species were purified from baculoviral/Sf9 expression and shown to bind essential and regulatory light chains. M. musculus myosin-18A, like the Drosophila melanogaster myosin-18s, has highly non-canonical motor properties. In actin-binding assays, both 18A and 18A bind weakly with Kd values of 4.6 and 41.9 M, respectively. Only 60% of myosin-18A bound to actin even at high actin concentrations, and only 25% of 18A bound, yet unbound motors could reassociate with actin with reiterated binding characteristics. This suggests an equilibrium between two conformations, competent and incompetent to bind actin, that we modeled with a two step process. Mant-nucleotides confirmed binding to motors, but neither 18A isoform hydrolyzed ATP despite either including smooth muscle tropomyosin, or phosphorylation of bound regulatory light chain, or addition of known binding partner GOLPH3 to the assay. Actin binding was unchanged by nucleotide. Negative stain electron microscopy confirmed the presence of one or two heads per molecule for S1 and HMM respectively. Alignment and classification of images of nucleotide free S1 revealed a molecule in which the lever is strongly angled with respect to the long axis of the motor.

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National Heart, Lung, and Blood Institute
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