The mitotic kinesin CENP-E is a processive transport motor

In vivo studies suggest that centromeric protein E (CENP-E), a kinesin-7 family member, plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. How CENP-E accomplishes this crucial task, however, is not clear. Here we present single-molecule measurements of CENP-E that demonstrate that this motor moves processively toward the plus end of microtubules, with an average run length of 2.6 +/- 0.2 mum, in a hand-over-hand fashion, taking 8-nm steps with a stall force of 6 +/- 0.1 pN. The ATP dependence of motor velocity obeys Michaelis-Menten kinetics with K(M,ATP) = 35 +/- 5 muM. All of these features are remarkably similar to those for kinesin-1-a highly processive transport motor. We, therefore, propose that CENP-E transports chromosomes in a manner analogous to how kinesin-1 transports cytoplasmic vesicles.     

Myosin VI is a processive motor with a large step size

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.     

A force-dependent state controls the coordination of processive myosin V

Myosin V is an efficient processive molecular motor. Recent experiments have shown how the structure and kinetics of myosin V are specialized to produce a highly processive motor capable of taking multiple 36-nm steps on an actin filament track. Here, we examine how two identical heads coordinate their activity to produce efficient hand-over-hand stepping. We have used a modified laser-trap microscope to apply a approximately 2-pN forward or backward force on a single-headed myosin V molecule, hypothesized to simulate forces experienced by the rear or lead head, respectively. We found that pulling forward produces only a small change in the kinetics, whereas pulling backward induces a large reduction in the cycling of the head. These results support a model in which the coordination of myosin V stepping is mediated by strain-generated inhibition of the lead head.     

Full-length myosin Va exhibits altered gating during processive movement on actin

Myosin Va (myoV) is a processive molecular motor that transports intracellular cargo along actin tracks with each head taking multiple 72-nm hand-over-hand steps. This stepping behavior was observed with a constitutively active, truncated myoV, in which the autoinhibitory interactions between the globular tail and motor domains (i.e., heads) that regulate the full-length molecule no longer exist. Without cargo at near physiologic ionic strength (100 mM KCl), full-length myoV adopts a folded (approximately 15 S), enzymatically-inhibited state that unfolds to an extended (approximately 11 S), active conformation at higher salt (250 mM). Under conditions favoring the folded, inhibited state, we show that Quantum-dot-labeled myoV exhibits two types of interaction with actin in the presence of MgATP. Most motors bind to actin and remain stationary, but surprisingly, approximately 20% are processive. The moving motors transition between a strictly gated and hand-over-hand stepping pattern typical of a constitutively active motor, and a new mode with a highly variable stepping pattern suggestive of altered gating. Each head of this partially inhibited motor takes longer-lived, short forward (35 nm) and backward (28 nm) steps, presumably due to globular tail-head interactions that modify the gating of the individual heads. This unique mechanical state may be an intermediate in the pathway between the inhibited and active states of the motor.     

Role of the lever arm in the processive stepping of myosin V

Myosin V is a two-headed molecular motor that binds six light chains per heavy chain, which creates unusually long lever arms. This motor moves processively along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed myosin V tightly binds to actin and swings its long lever arm through a large angle, providing a stroke. We created single-headed constructs with different-size lever arms and show that stroke size is proportional to lever arm length. In a two-headed molecule, the stroke provides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processive step seen in tissue-purified myosin V. Two-headed myosin V molecules with only four light chains per head are still processive, but their step size is reduced to 24 nm. A further reduction in the length of the lever arms to one light chain per head results in a motor that is unable to walk processively. This motor produces single small approximately 6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a given binding event. These data show that for myosin V with its normal proximal tail domain, both heads and a long lever arm are required for large, processive steps.     

Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.

The human HELB protein was first identified as a homolog of a putative murine replicative helicase (1–3). Since then, various functions have been assigned to the protein, including a role in the onset of chromosomal DNA replication (2), cellular recovery from replication stress (4), promotion of Cdc45 chromatin binding (5), resolution of DNA secondary CGG nucleotides repeat structures (6), and stimulation of RAD51-mediated 5′–3′ heteroduplex extension to promote homologous recombination (HR) (7). Most recently and in apparent contradiction to the role in the stimulation of HR, HELB was proposed to inhibit homology-dependent double-stranded DNA break (DSB) repair by antagonizing the processive resection nucleases EXO1 and DNA2/BLM during the G0/G1 phases of the cell cycle (8). In agreement with this idea, HELB forms nuclear foci in response to DNA damage and is phosphorylated by cyclin-dependent kinase (CDK), causing localization to the nucleus in G1 and to the cytoplasm during S/G2. The formation of HELB damage foci is dependent on the main eukaryotic single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) (9), which has been shown to interact physically with HELB (4, 8). Although the interaction with RPA is potentially critical for all putative functions of HELB, the ability of this motor protein to modulate the formation, remodeling, or removal of RPA nucleoprotein filaments has never been studied and is the focus of the work presented here.The filaments formed between RPA and ssDNA are critical intermediates in DNA replication, recombination, and repair (10–12). RPA not only shields ssDNA from nucleolytic degradation, but it is also involved in the recruitment or exclusion of other factors from ssDNA, the regulation of DNA replication and repair, and the initiation of cell signaling cues that link these pathways to the cell cycle and its progression through checkpoints (13). Interestingly, many helicases and helicase-like proteins share intimate physical and functional interactions with ssDNA binding proteins (14, 15). However, we do not currently understand how the activity of HELB affects RPA filaments and vice versa.HELB is a 120-kDa protein comprising three distinct domains: an N-terminal region of unknown function, a central helicase domain sharing homology with the Superfamily 1 (SF1) helicase RecD, and a C-terminal region containing CDK phosphorylation sites (3) (Fig. 1A). Site-directed mutagenesis has implicated the central helicase domain in both the DNA and RPA binding activities of HELB (Fig. 1A, blue arrows). Interestingly, mutations in HELB are associated with both female infertility and early-onset menopause and are found widely distributed in the protein sequence in human tumor samples (Fig. 1A, red arrows) (16, 17). In vitro studies show that HELB possesses ssDNA-dependent ATPase activity and 5′ to 3′ helicase activity, which is as expected based on the similarity to RecD (2, 18). However, precisely how these biochemical properties underpin the cellular function(s) of HELB and the significance of the interaction with RPA are unresolved.Open in a separate windowFig. 1.HELB is a monomer that binds tightly to ssDNA and displays ssDNA-dependent ATPase activity. (A) Cartoon of HELB showing overall domain layout and important mutations. Red marks denote positions of high-frequency tumor mutations. Blue marks denote positions of mutations that affect ATPase, DNA binding, and RPA binding activities. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis shows highly purified recombinant human HELB produced from insect cells. (C) SEC-MALS analysis demonstrates that HELB is a monomer in solution under these conditions with a calculated molecular mass (red data line) of 123,252 Da. (D, left axis) HELB binding constants (K_d) for poly(dT) substrates of different lengths obtained in PIFE assays described in SI Appendix, Fig. S1A. An exponential fit determines a saturating K_d of 5 nM. (D, right axis) Stoichiometry values obtained under tight binding conditions as shown in SI Appendix, Fig. S1 B–E. (E) Michaelis–Menten plot of ATP hydrolysis gives K_m and k_cat parameters for WT HELB and also shows that the K481A mutant is unable to hydrolyze ATP. (F) Analysis of DNA stimulation of HELB ATPase activity demonstrates that HELB is an ssDNA-dependent helicase. ATP turnover is stimulated more by polythymidine substrates than mixed base sequences, likely due to their inability to form inhibitory secondary structures. A.U., arbitrary units; mw, molecular weight.In this study, we used bulk and single-molecule assays to further characterize HELB, including its physical and functional interactions with RPA and RPA nucleoprotein filaments. Paradoxically, we find that human RPA (itself a potent ssDNA binding protein) is stimulatory to all activities of HELB on ssDNA, despite the competition one would expect between the two proteins for their nucleic acid substrates. In contrast, noncognate RPA protein inhibits all activities of human HELB. These highly specific interactions with RPA filaments help to recruit HELB onto ssDNA that is devoid of secondary structure and promote efficient ssDNA translocation coupled to the processive clearance of RPA. The implications of this finding for the roles of HELB in DNA replication and recombination are discussed.     

A FERM domain autoregulates Drosophila myosin 7a activity

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V_max ≈ 1s⁻¹ and K_ATPase ≈ 1 μM actin. We find that myosin 7a-FL has V_max similar to S1 but K_ATPase ≈ 30 μM. Thus, at low actin concentrations (5 μM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K_ATPase is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K_ATPase to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K_d ≈ 30 μM regardless of ATP, similar to the K_ATPase value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.     

A conventional myosin motor drives neurite outgrowth

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actin-associated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.     

Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner

Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but heterodimerization with a processive partner generates processivity. The “unprocessive” subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.     

Localization of a myosin heavy chain-like polypeptide to Drosophila nuclear pore complexes.

Antibodies previously used for immunofluorescence localization of a myosin heavy chain-like polypeptide to the nuclear envelope in higher eukaryotic cells crossreact with both muscle and nonmuscle isoforms of Drosophila myosin heavy chain. Analyses of Drosophila tissue culture cells and premyogenic embryos suggest that it is the nonmuscle isoform that is associated with the nuclear envelope. Further immunofluorescence and immunoelectron microscopy indicate that this polypeptide is associated with nuclear pore complexes. These data support the hypothesis put forward previously that myosin or myosin-like molecules play a role in pore complex architecture.     

Kinetic characterization of a cytoplasmic myosin motor domain expressed in Dictyostelium discoideum.

A detailed kinetic study of the interaction of a recombinant myosin head fragment (MHF) of Dictyostelium discoideum with actin and adenine nucleotides has been made by using a combination of rapid-reaction, equilibrium, and fluorescence methods. MHF is equivalent in size to a proteolytic fragment of skeletal muscle myosin, subfragment 1 (S1), the simplest unit of myosin to retain enzymatic and functional activity. The results show that qualitatively the interactions of MHF with nucleotides and actin are the same as those of S1. Both bind to rabbit actin with the same affinity, although differences in the rate constants of their interactions with nucleotides in the presence and absence of actin occur. The rate of ATP binding to MHF and the subsequent cleavage step are significantly slower than the corresponding rates with S1. The dissociation of a fluorescent analog of ADP from MHF was 5-fold faster than from S1, while its rate of binding MHF was 3-fold slower, resulting in a weaker association equilibrium constant. The ATP-induced isomerization of the actoMHF complex was 10-fold slower than for actoS1, but the binding affinities of ADP for actoMHF and actoS1 were indistinguishable. The results suggest a different degree of coupling between the nucleotide and actin binding sites of MHF and S1 which may be a common feature of nonmuscle myosins. They also provide the basis for a study of specifically modified myosins with which one can probe the sites of interaction with nucleotides or actin, as well as functional motility.     

A myosin motor that selects bundled actin for motility

Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.     

Cargo binding activates myosin VIIA motor function in cells

Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.     

Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa,the Usher syndrome 1B protein

Mutations in the myosin VIIa gene (MYO7A) cause Usher syndrome type 1B (USH1B), a major type of the deaf-blind disorder, Usher syndrome. We have studied mutant phenotypes in the retinas of Myo7a mutant mice (shaker1), with the aim of elucidating the role(s) of myosin VIIa in the retina and what might underlie photoreceptor degeneration in USH1B patients. A photoreceptor defect has been described. Here, we report that the phagocytosis of photoreceptor outer segment disks by the retinal pigment epithelium (RPE) is abnormal in Myo7a null mice. Both in vivo and in primary cultures of RPE cells, the transport of ingested disks out of the apical region is inhibited in the absence of Myo7a. The results with the cultured RPE cells were the same, irrespective of whether the disks came from wild-type or mutant mice, thus demonstrating that the RPE is the source of this defect. The inhibited transport seems to delay phagosome-lysosomal fusion, as the degradation of ingested disks was slower in mutant RPE. Moreover, fewer packets of disk membranes were ingested in vivo, possibly because retarded removal of phagosomes from the apical processes inhibited the ingestion of additional disk membranes. We conclude that Myo7a is required for the normal processing of ingested disk membranes in the RPE, primarily in the basal transport of phagosomes into the cell body where they then fuse with lysosomes. Because the phagocytosis of photoreceptor disks by the RPE has been shown to be critical for photoreceptor cell viability, this defect likely contributes to the progressive blindness in USH1B.     

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin–specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end–directed motor that moves actin filaments in a gliding assay (∼430 nm·s⁻¹ at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k_cat ∼6 s⁻¹) was similar to the actin-detachment rate (k_det = 6.2 s⁻¹) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.Unconventional myosin 15 is expressed by inner ear hair cells and accumulates at the tips of mechanosensory stereocilia (1, 2), actin-based organelles that are stimulated by sound and accelerations. A missense substitution in the myosin 15 motor domain results in abnormally short stereocilia in the Myo15^sh2/sh2 (shaker 2) mouse, indicating that myosin 15 motor activity is essential for elongation of the core actin filaments during development (1, 3). Emphasizing the critical importance of myosin 15 for sensory function, mutations of the orthologous MYO15A also cause profound nonsyndromic autosomal recessive deafness (DFNB3) in humans (4, 5). Myosin 15 is hypothesized to be a molecular motor that transports the scaffolding protein whirlin (DFNB31) and epidermal growth factor receptor kinase substrate 8 (EPS8) to the stereocilia tips, where these cargos regulate elongation of the developing actin core (6, 7). Although myosin 15 motility has not been visualized in live hair cells, it similarly transports whirlin and EPS8 to the tips of filopodia, finger-like protrusions of bundled actin filaments (6, 7). Little is known about the functional properties of myosin 15, its mechanism of motility along stereocilia or filopodial actin filaments, or how this might be altered by mutations associated with DFNB3 human deafness.Myosins are a superfamily of actin-activated P-loop ATPases that produce force to power fundamental cellular processes, such as cytokinesis and vesicle trafficking (8). Biochemical and biophysical analyses of purified isozymes have been critical to deciphering their cellular functions. Most myosins share a common catalytic mechanism, but kinetic tuning of reaction rates can result in very different characteristics (9, 10). An important property is the duty ratio; defined as the fraction of the ATPase cycle that myosin spends strongly bound to actin. Low-duty ratio motors, exemplified by muscle myosins, spend a small fraction (∼0.05) of their cycle strongly bound to actin and act in large ensembles. In contrast, processive motors like myosin 5a have a duty ratio of >0.7 at saturating [actin], and thus spend most of their ATPase cycle in strongly actin-bound states (11). For myosin 5a, the high-duty ratio supports the processive movement of dimers by increasing the likelihood that one head is always bound to actin, thus preventing diffusion away from the filament track.Myosin 15 is the largest myosin heavy chain in the mammalian proteome and is highly expressed in the inner ear and endocrine organs (2). Phylogenetic analysis of motor domain sequences reveals its close relationship with unconventional class VII and X myosins, in addition to sharing similar myosin tail homology 4 (MyTH4), Src homology 3 (SH3) and band 4.1, ezrin, radixin, moesin (FERM) domains in the C-terminal tail (Fig. 1A). Alternate splicing of exon 2 (1, 2), creates two protein isoforms that are identical except for a large (133 kDa) proline-rich N-terminal extension that is unique to isoform 1 (Fig. 1A). The function of isoform 1 is unknown, whereas isoform 2 targets to stereocilia and filopodia tips, and is sufficient to rescue stereocilia development in Myo15^sh2/sh2 hair cells (1, 6). Myosin 15 has three presumed light-chain binding sites (IQ motifs) that are predicted to form a lever arm; however, the light chains that potentially bind these in vivo are unknown.Open in a separate windowFig. 1.Purification of mouse myosin 15 from Sf9 cells by chaperone coexpression. (A) Schematic domain structure of mouse myosin 15 isoforms. (B) Motor domain constructs used in this study. All expressed proteins have a C-terminal FLAG epitope for affinity purification. (C) ClustalW alignments of the second IQ of myosin 15 show similarity to the ELC binding site (first IQ) of class II myosins. (D) Coexpression with UNC45B/HSP90AA1 increases the solubility of EGFP-Myo15-3IQ in Sf9 insect cells (abbreviated to 3IQ for D–F). Western blot probed with antibody against FLAG (green) and alpha-tubulin (red). Total and soluble fractions were prepared from equal volumes of Sf9 cell cultures expressing EGFP-Myo15-3IQ, plus combinations of chaperones and light chains as shown. EGFP-Myo15-3IQ sedimented when coexpressed with calmodulin (CALM) alone, or with CALM + ELC + RLC. Expression of UNC45B + HSP90AA1 increased EGFP-Myo15-3IQ in the supernatant (compare arrowheads), but did not change heavy chain expression overall (compare stars). (E) Supernatants from D were bound to FLAG-M2 affinity resin, eluted with FLAG peptide (0.5-mL fractions), and assayed for EGFP fluorescence (representative of three experiments). Individual images (outlined in white) for each condition were captured with identical settings and consolidated. Samples coexpressing RLC + ELC in addition to UNC45B + HSP90AA1 eluted in earlier fractions (third row), compared with coexpressing UNC45B/HSP90AA1 and CALM (second row). (F) SDS/PAGE of FLAG-purified proteins from Sf9 cells expressing UNC45B + HSP90AA1 and combinations of Myo15 IQ mutants and lights chains as indicated. Note increased mobility of Myo15-1IQ (arrowhead) without an EGFP fusion.To understand how myosin 15 operates within the stereocilia organelle, we have purified the motor domain and used a combination of single molecule and ensemble kinetic/mechanical analyses to determine its properties. While attempting to purify myosin 15 from Spodoptera frugiperda (Sf9) insect cells, we discovered that coexpression of heat-shock protein 90 (HSP90) and its cochaperone UNC45B significantly improved the yield of soluble protein. We demonstrate that purified myosin 15 is a mechanically active, barbed-end–directed molecular motor that exhibits a high-duty ratio. These data suggest that myosin 15 might be capable of processive motility if dimerized, consistent with its proposed role as a stereocilia transporter.     

Structural mechanism of the recovery stroke in the myosin molecular motor

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ( approximately 60 degrees ) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm.