Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.ATPase ͉ electron microscopy ͉ optical trap ͉ single alpha helix M yosins form a superfamily of motor proteins, which are ubiquitous and responsible for a wide range of movement in cells. They all contain three basic components: (i) a motor domain that binds actin and hydrolyzes ATP to generate force, (ii) a lever that contains ''IQ motifs'' to which light chains (generally calmodulin) bind, and (iii) a tail, which directs the cellular function of each myosin. There are 12 classes of myosins in the human genome, of which 11 are the so-called ''unconventional myosins'' (1, 2). One of the best characterized of the unconventional myosins is myosin 5a (Myo5). This dimeric myosin has a long lever (six IQ motifs), which allows it to bind to sites on actin 36 nm apart (the spacing of the actin filament helical pseudo-repeat) and thus ''walk'' straight along actin, taking 36-nm steps (3, 4). Its high duty ratio (3-5) enhances its ability to take several steps along actin before falling off (i.e., to behave processively).We recently challenged the convention that the myosin lever always consists solely of IQ motifs plus their light chains. We showed that the predicted coiled-coil domain of myosin 10 actually forms a stable single ␣-helical (SAH) domain, using a synthetic peptide (6). SAH domains lack the hydrophobic seam found in coiled-coil ␣-helices, and are highly charged (rich in E, K, and R residues), with many (i, i Ϯ 4) and (i, i Ϯ 3) stabilizing intrahelical ionic bonds between either K and E, or R and E (2). We further sh...

Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/ extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s −1 ) is similar to the maximum ATPase activity (∼12-14 s −1 ) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircaselike processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.actomyosin | stable single alpha-helix | optical trapping | myosin X | myosin-5a

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