The M. musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, maintenance of cytoskeleton, and as 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. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant HMM- and S1-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains indicating an additional noncanonical light chaing binding site in the neck. Myosin-18Aα and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 uM, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound mant-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002s-1) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis and neither did addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-powerstroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.
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