Manuel H. Taft

The mechanism of pentabromopseudilin inhibition of myosin motor activity

We have identified pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and unloaded shortening velocity. PBP-induced reductions in the rate constants for ATP binding, ATP hydrolysis and ADP dissociation extend the time required per myosin ATPase cycle in the absence and presence of actin. Additionally, coupling between the actin and nucleotide binding sites is reduced in the presence of the inhibitor. The selectivity of PBP differs from that observed with other myosin inhibitors. To elucidate the binding mode of PBP, we crystallized the Dictyostelium myosin-2 motor domain in the presence of Mg2+-ADP–meta-vanadate and PBP. The electron density for PBP is unambiguous and shows PBP to bind at a previously unknown allosteric site near the tip of the 50-kDa domain, at a distance of 16 Å from the nucleotide binding site and 7.5 Å away from the blebbistatin binding pocket.

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

Small molecule-mediated refolding and activation of myosin motor function

The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001

Crystal Structure of Human Myosin 1c—The Motor in GLUT4 Exocytosis: Implications for Ca2 + Regulation and 14-3-3 Binding

Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca(2+) binding. Contact residues in the motor domain are linked to the central β-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca(2+) binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3β (KD=0.9 μM) and present the model of a double-headed Myo1c-14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.

Dictyostelium Myosin-5b Is a Conditional Processive Motor

Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg2+-ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg2+-ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.

Functional characterization of human myosin-18A and its interaction with F-actin and GOLPH3.

Background: Class-18A myosins share a unique N-terminal extension comprising a PDZ module and a KE-rich region. Results: Human myosin-18A binds F-actin via its motor domain in a nucleotide-dependent manner and via the KE-rich region, modulated by direct interaction between the PDZ module and GOLPH3. Conclusion: Myosin-18A binds F-actin and recruits interaction partners to the cytoskeleton. Significance: This work establishes a molecular basis for myosin-18A mediated membrane-cytoskeleton interplay. Molecular motors of the myosin superfamily share a generic motor domain region. They commonly bind actin in an ATP-sensitive manner, exhibit actin-activated ATPase activity, and generate force and movement in this interaction. Class-18 myosins form heavy chain dimers and contain protein interaction domains located at their unique N-terminal extension. Here, we characterized human myosin-18A molecular function in the interaction with nucleotides, F-actin, and its putative binding partner, the Golgi-associated phosphoprotein GOLPH3. We show that myosin-18A comprises two actin binding sites. One is located in the KE-rich region at the start of the N-terminal extension and appears to mediate ATP-independent binding to F-actin. The second actin-binding site resides in the generic motor domain and is regulated by nucleotide binding in the absence of intrinsic ATP hydrolysis competence. This core motor domain displays its highest actin affinity in the ADP state. Electron micrographs of myosin-18A motor domain-decorated F-actin filaments show a periodic binding pattern independent of the nucleotide state. We show that the PDZ module mediates direct binding of myosin-18A to GOLPH3, and this interaction in turn modulates the actin binding properties of the N-terminal extension. Thus, myosin-18A can act as an actin cross-linker with multiple regulatory modulators that targets interacting proteins or complexes to the actin-based cytoskeleton.

Myosin-1C associates with microtubules and stabilizes the mitotic spindle during cell division

The mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin–MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and tail-domain-mediated MT–F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.

Load-dependent modulation of non-muscle myosin-2A function by tropomyosin 4.2

Tropomyosin isoforms play an important role in the organisation of cytoplasmic actomyosin complexes in regard to function and cellular localisation. In particular, Tpm4.2 is upregulated in rapidly migrating cells and responsible for the specific recruitment of the cytoplasmic class-2 myosin NM-2A to actin filaments during the formation of stress fibres. Here, we investigate how the decoration of F-actin with Tpm4.2 affects the motor properties of NM-2A under conditions of low and high load. In the absence of external forces, decoration of actin filaments with Tpm4.2 does not affect the gated release of ADP from NM-2A and the transition from strong to weak actin-binding states. In the presence of resisting loads, our results reveal a marked increase in the mechanosensitive gating between the leading and trailing myosin head. Thereby, the processive behaviour of NM-2A is enhanced in the presence of resisting loads. The load- and Tpm4.2-induced changes in the functional behaviour of NM-2A are in good agreement with the role of this myosin in the context of stress fibres and the maintenance of cellular tension.

Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin.

Phalloidin and fluorescently labeled phalloidin analogs are established reagents to stabilize and mark actin filaments for the investigation of acto‐myosin interactions. In the present study, we employed transient and steady‐state kinetic measurements as well as in vitro motility assays to show that phalloidin perturbs the productive interaction of human non‐muscle myosin‐2A and ‐2C1 with filamentous actin. Phalloidin binding to F‐actin results in faster dissociation of the complex formed with non‐muscle myosin‐2A and ‐2C1, reduced actin‐activated ATP turnover, and slower velocity of actin filaments in the in vitro motility assay. In contrast, phalloidin binding to F‐actin does not affect the interaction with human non‐muscle myosin isoform 2B and Dictyostelium myosin‐2 and myosin‐5b.