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Effective tissue repair after myocardial infarction entails a vigorous angiogenic response, guided by incompletely defined immune cell–endothelial cell interactions. We identify the monocyte- and macrophage-derived cytokine METRNL (meteorin-like) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT (KIT receptor tyrosine kinase). METRNL mediated angiogenic effects in cultured human endothelial cells through KIT-dependent signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl -deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue repair.

KEY POINTS A nanomachine made of an ensemble of seven heavy-meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half-sarcomere generating steady force and constant-velocity shortening. To preserve Ca2+ as a free parameter, the Ca2+ -insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser-trapped bead acting as a force transducer. The new method reveals that the performance of the nanomachine powered by myosin from frog hind-limb muscles depends on [Ca2+ ], an effect mediated by a Ca2+ -binding site in the regulatory light chain of HMM. The Ca2+ -sensitivity is class-specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca2+ independent. A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin. ABSTRACT An ensemble of seven heavy-meromyosin (HMM) fragments of myosin-II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca2+ -insensitive gelsolin fragment TL40, making [Ca2+ ] a free parameter. Frog myosin performance in 2 mm ATP is affected by Ca2+ : in 0.1 mm Ca2+ , the isometric steady force (F0 , 15.25 pN) is increased by 50% (P = 0.004) with respect to that in Ca2+ -free solution, the maximum shortening velocity (V0 , 4.6 μm s-1 ) is reduced by 27% (P = 0.46) and the maximum power (Pmax , 7.6 aW) is increased by 21% (P = 0.17). V0 reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%, P

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein–protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0–C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.

A novel cytoplasmic dye decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds and general peroxidase substrates like ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.

A novel cytoplasmic dye decolorizing peroxidase from Dictyostelium discoideum was investigated for its activity towards lignin oxidation. In contrast to related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer and oxidizes anthraquinone dyes, lignin model compounds and general peroxidase substrates like ABTS efficiently. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the distal and a water molecule at the axial face. Asp149 is in an optimal position to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long range electron transfer pathways associated with a hydrogen bonding network that connects the substrate binding sites with the heme moiety are described.

Key points Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of β-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the β-cardiac myosin-2 motor domain with an EC50 of 200 nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. Abstract Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine β-cardiac myosin, the EC50 for rumenic acid varies from 10.5 μM in the absence of actin to 200 nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of β-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5 μM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

A nanomachine made of an ensemble of seven heavy-meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half-sarcomere generating steady force and constant-velocity shortening. To preserve CaAn ensemble of seven heavy-meromyosin (HMM) fragments of myosin-II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca

Class XVIII myosins represent a branch of the myosin family tree characterized by the presence of large N- and C-terminal extensions flanking a generic myosin core. These myosins display the highest sequence similarity to conventional class II muscle myosins and are compatible with but not restricted to myosin-2 contractile structures. Instead, they fulfill their functions at diverse localities, such as lamella, actomyosin bundles, the Golgi apparatus, focal adhesions, the cell membrane, and within sarcomeres. Sequence comparison of active-site residues and biochemical data available thus far indicate that this myosin class lacks active ATPase-driven motor activity, suggesting that its members function as structural myosins. An emerging body of evidence indicates that this structural capability is essential for the organization, maturation, and regulation of the contractile machinery in both muscle and nonmuscle cells. This is supported by the clear association of myosin-18A (Myo18A) and myosin-18B (Myo18B) dysregulation with diseases such as cancer and various myopathies.

Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.

Germline mutations in the ubiquitously expressed ACTB, which encodes β-cytoplasmic actin (CYA), are almost exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF). Here, we report six patients with previously undescribed heterozygous variants clustered in the 3′-coding region of ACTB. Patients present with clinical features distinct from BWCFF, including mild developmental disability, microcephaly, and thrombocytopenia with platelet anisotropy. Using patient-derived fibroblasts, we demonstrate cohort specific changes to β-CYA filament populations, which include the enhanced recruitment of thrombocytopenia-associated actin binding proteins (ABPs). These perturbed interactions are supported by in silico modeling and are validated in disease-relevant thrombocytes. Co-examination of actin and microtubule cytoskeleton constituents in patient-derived megakaryocytes and thrombocytes indicates that these β-CYA mutations inhibit the final stages of platelet maturation by compromising microtubule organization. Our results define an ACTB-associated clinical syndrome with a distinct genotype-phenotype correlation and delineate molecular mechanisms underlying thrombocytopenia in this patient cohort. Genetic variants in ACTB and ACTG1 have been associated with Baraitser-Winter Cerebrofrontofacial syndrome. Here, the authors report of a syndromic thrombocytopenia caused by variants in ACTB exons 5 or 6 that compromise the organization and coupling of the cytoskeleton, leading to impaired platelet maturation.

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.

The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear–cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5-fold more than the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C35 (NTR35) docked to the X-ray structure of MYO1CC, we predicted that MYO1C35 NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35, which harbors the R21G mutation, was unable to confer MYO1C35-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.

The cyanoacrylate compound phenamacril (also known as JS399-19) is a recently identified fungicide that exerts its antifungal effect on susceptible Fusarium species by inhibiting the ATPase activity of their myosin class I motor domains. Although much is known about the antifungal spectrum of phenamacril, the exact mechanism behind the phenamacril-mediated inhibition remains to be resolved. Here, we describe the characterization of the effect of phenamacril on purified myosin motor constructs from the model plant pathogen and phenamacril-susceptible species Fusarium graminearum, phenamacril-resistant Fusarium species, and the mycetozoan model organism Dictyostelium discoideum Our results show that phenamacril potently (IC 50 ∼360 nm), reversibly, and noncompetitively inhibits ATP turnover, actin binding during ATP turnover, and motor activity of F. graminearum myosin-1. Phenamacril also inhibits the ATPase activity of Fusarium avenaceum myosin-1 but has little or no inhibitory effect on the motor activity of Fusarium solani myosin-1, human myosin-1c, and D. discoideum myosin isoforms 1B, 1E, and 2. Our findings indicate that phenamacril is a species-specific, noncompetitive inhibitor of class I myosin in susceptible Fusarium sp.

International journal of molecular sciences 22(12), 6265 (2021). doi:10.3390/ijms22126265, A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 �� resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN��� molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described., Published by Molecular Diversity Preservation International, Basel

The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.

MYO18B loss-of-function mutations and depletion significantly compromise the structural integrity of striated muscle sarcomeres. The molecular function of the encoded protein, myosin-18B (M18B), within the developing muscle is unknown. Here, we demonstrate that recombinant M18B lacks motor ATPase activity and harbors previously uncharacterized N-terminal actin-binding domains, properties that make M18B an efficient actin cross-linker and molecular brake capable of regulating muscle myosin-2 contractile forces. Spatiotemporal analysis of M18B throughout cardiomyogenesis and myofibrillogenesis reveals that this structural myosin undergoes nuclear-cytoplasmic redistribution during myogenic differentiation, where its incorporation within muscle stress fibers coincides with actin striation onset. Furthermore, this analysis shows that M18B is directly integrated within the muscle myosin thick filament during myofibril maturation. Altogether, our data suggest that M18B has evolved specific biochemical properties that allow it to define and maintain sarcomeric organization from within the thick filament via its dual actin cross-linking and motor modulating capabilities.

The cyanoacrylate compound phenamacril (also known as JS399–19) is a recently identified fungicide that exerts its antifungal effect on susceptible Fusarium species by inhibiting the ATPase activity of their myosin class I motor domains. Although much is known about the antifungal spectrum of phenamacril, the exact mechanism behind the phenamacril-mediated inhibition remains to be resolved. Here, we describe the characterization of the effect of phenamacril on purified myosin motor constructs from the model plant pathogen and phenamacril-susceptible species Fusarium graminearum, phenamacril-resistant Fusarium species, and the mycetozoan model organism Dictyostelium discoideum. Our results show that phenamacril potently (IC(50) ∼360 nm), reversibly, and noncompetitively inhibits ATP turnover, actin binding during ATP turnover, and motor activity of F. graminearum myosin-1. Phenamacril also inhibits the ATPase activity of Fusarium avenaceum myosin-1 but has little or no inhibitory effect on the motor activity of Fusarium solani myosin-1, human myosin-1c, and D. discoideum myosin isoforms 1B, 1E, and 2. Our findings indicate that phenamacril is a species-specific, noncompetitive inhibitor of class I myosin in susceptible Fusarium sp.

Introductory paragraphUntil recently missense germ-line mutations inACTB, encoding the ubiquitously expressed β-cytoplasmic actin (CYA), were exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF), a complex developmental disorder1,2. Here, we report six patients with previously undescribed heterozygous variants clustered in the 3’-coding region ofACTB. These patients present with clinical features different from BWCFF, including thrombocytopenia, microcephaly, and mild developmental disability. Patient derived cells are morphologically and functionally distinct from controls. Assessment of cytoskeletal constituents identified a discrete filament population altered in these cells, which comprises force generating and transmitting actin binding proteins (ABP) known to be associated with thrombocytopenia3–8.In silicomodelling and molecular dynamics (MD)-simulations support altered interactions between these ABP and mutant β-CYA. Our results describe a new clinical syndrome associated withACTBmutations with a distinct genotype-phenotype correlation, identify a cytoskeletal protein interaction network crucial for thrombopoiesis, and provide support for the hypomorphic nature of these actinopathy mutations.

Tropomyosin proteins form stable coiled-coil dimers that polymerize along the α-helical groove of actin filaments [1]. The actin cytoskeleton consists of both co-polymers of actin and tropomyosin and polymers of tropomyosin-free actin [2]. The fundamental distinction between these two types of filaments is that tropomyosin determines the functional capability of actin filaments in an isoform-dependent manner [3-9]. However, it is unknown what portion of actin filaments are associated with tropomyosin. To address this deficit, we have measured the relative distribution between these two filament populations by quantifying tropomyosin and actin levels in a variety of human cell types, including bone (U2OS); breast epithelial (MCF-10A); transformed breast epithelial (MCF-7); and primary (BJpar), immortalized (BJeH), and Ras-transformed (BJeLR) BJ fibroblasts [10]. Our measurements of tropomyosin and actin predict the saturation of the actin cytoskeleton, implying that tropomyosin binding must be inhibited in order to generate tropomyosin-free actin filaments. We find the majority of actin filaments to be associated with tropomyosin in four of the six cell lines tested and the portion of actin filaments associated with tropomyosin to decrease with transformation. We also discover that high-molecular-weight (HMW), unlike low-molecular-weight (LMW), tropomyosin isoforms are primarily co-polymerized with actin in untransformed cells. This differential partitioning of tropomyosins is not due to a lack of N-terminal acetylation of LMW tropomyosins, but it is, in part, explained by the susceptibility of soluble HMW tropomyosins to proteasomal degradation. We conclude that actin-tropomyosin co-polymers make up a major fraction of the human actin cytoskeleton.

The metazoan actin cytoskeleton supports a wide range of contractile and transport processes. Recent studies have shown how the dynamic association with specific tropomyosin isoforms generates actin filament populations with distinct functional properties. However, critical details of the associated molecular interactions remain unclear. Here, we report the properties of actomyosin–tropomyosin complexes containing filamentous β-actin, nonmuscle myosin-2B (NM-2B) constructs, and either tropomyosin isoform Tpm1.8cy (b.–.b.d), Tpm1.12br (b.–.b.c), or Tpm3.1cy (b.–.a.d). Our results show the extent to which the association of filamentous β-actin with these different tropomyosin cofilaments affects the actin-mediated activation of NM-2B and the release of the ATP hydrolysis products ADP and phosphate from the active site. Phosphate release gates a transition from weak to strong F-actin–binding states. The release of ADP has the opposite effect. These changes in dominant rate-limiting steps have a direct effect on the duty ratio, the fraction of time that NM-2B spends in strongly F-actin–bound states during ATP turnover. The duty ratio is increased ∼3-fold in the presence of Tpm1.12 and 5-fold for both Tpm1.8 and Tpm3.1. The presence of Tpm1.12 extends the time required per ATP hydrolysis cycle 3.7-fold, whereas it is shortened by 27 and 63% in the presence of Tpm1.8 and Tpm3.1, respectively. The resulting Tpm isoform–specific changes in the frequency, duration, and efficiency of actomyosin interactions establish a molecular basis for the ability of these complexes to support cellular processes with widely divergent demands in regard to force production, capacity to move processively, and speed of movement.

The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear–cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5-fold more than the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C35 (NTR35) docked to the X-ray structure of MYO1CC, we predicted that MYO1C35 NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35, which harbors the R21G mutation, was unable to confer MYO1C35-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.

Myosin motor proteins convert chemical energy into force and movement through their interactions with nucleotide and filamentous actin (F-actin). The evolutionarily conserved lysine-265 (K265) of the myosin-2 motor from Dictyostelium discoideum (Dd) is proposed to be a key residue in an allosteric communication pathway that mediates actin-nucleotide coupling. To better understand the role of K265, point mutations were introduced within the Dd myosin-2 M765-2R framework, replacing this lysine with alanine (K265A), glutamic acid (K265E) or glutamine (K265Q), and the functional and kinetic properties of the resulting myosin motors were assessed. The alanine and glutamic acid substitutions reduced actin-activated ATPase activity, slowed the in vitro sliding velocity and attenuated the inhibitory potential of the allosteric myosin inhibitor pentabromopseudilin (PBP). However, glutamine substitution did not substantially change these parameters. Structural modelling suggests that K265 interacts with D590 and Q633 to establish a pivotal allosteric branching point. Based on our results, we propose: (1) that the K265-D590 interaction functions to reduce myosins basal ATPase activity in the absence of F-actin, and (2) that the dynamic formation of the K265-Q633 salt bridge upon actin cleft closure regulates the activation of product release by actin filaments.

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.

Mutation-induced dysfunction or misfolding of heart muscle sarcomere components have been linked to dilated and hypertrophic cardiomyopathies. We have previously shown that the small molecule EMD57033 increases β-cardiac myosin activity and acts as a pharmacological chaperone that stabilizes and refolds the motor domain. Following up on this finding, we investigated whether metabolites such as fatty acids can have a similar effect on β-cardiac myosin. Arachidonic acid (AA) was previously reported as an activator of smooth muscle myosin. We found that among all fatty acids tested, AA induced the largest effect on actin-activated ATPase activity of β-cardiac myosin. Closer characterization revealed a 13-fold increase in β-cardiac myosins affinity for actin in the presence of ATP and a 7-fold increase in the rate of phosphate release. We further aimed to elucidate the effect of AA-induced myosin activation in the context of a reconstituted human cardiac actin-troponin-tropomyosin complex. Both, assays monitoring ATPase and in vitro motility showed a significant increase in activity over a wide range of Ca2+ concentrations. Similar to the effect of EMD57033, the addition of AA increases the stability of myosin and reverses the loss of myosin activity caused by temperature stress. The refolding reaction can be monitored by the recovery of the intrinsic protein fluorescence signal that is associated with ATP binding and active turnover. Alternatively, we followed the clear reduction in the number of immobile filaments following the addition of AA.

Septin 7 (SEPT7) has been described to be essential for successful completion of cytokinesis in mouse fibroblasts and Sept7-deficiency in fibroblasts constitutively results in multinucleated cells which stop proliferation. Using Sept7flox/floxfibroblasts we generated a cellular system, where the cytokinetic defects of Cre-mediated deletion of the Sept7 gene can be rescued by ectopically expressed doxycycline-inducible wild type SEPT7. Using this system, we analyzed the ability of SEPT7-mutants with alterations in their GTPase domain-dependent dimerization to prevent multinucleation and rescue proliferation. Although biochemical analysis of the mutants demonstrates differences in homo- and/or hetero-polymerization, in GTP-binding and/or GTPase activities, all analyzed mutants were able to rescue the cytokinesis phenotype of Sept7flox/floxfibroblasts associated with Cre-mediated deletion of endogenous Sept7. These findings indicate that the ability of septins to assemble into well-defined SEPT7-dimerization dependent native filaments is dispensable for cytokinesis in fibroblasts and opens the way to search for other mechanisms of the involvement of SEPT7 in cytokinesis.

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.

Germline mutations in the ubiquitously expressed ACTB, which encodes β-cytoplasmic actin (CYA), are almost exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF). Here, we report six patients with previously undescribed heterozygous variants clustered in the 3′-coding region of ACTB. Patients present with clinical features distinct from BWCFF, including mild developmental disability, microcephaly, and thrombocytopenia with platelet anisotropy. Using patient-derived fibroblasts, we demonstrate cohort specific changes to β-CYA filament populations, which include the enhanced recruitment of thrombocytopenia-associated actin binding proteins (ABPs). These perturbed interactions are supported by in silico modeling and are validated in disease-relevant thrombocytes. Co-examination of actin and microtubule cytoskeleton constituents in patient-derived megakaryocytes and thrombocytes indicates that these β-CYA mutations inhibit the final stages of platelet maturation by compromising microtubule organization. Our results define an ACTB-associated clinical syndrome with a distinct genotype-phenotype correlation and delineate molecular mechanisms underlying thrombocytopenia in this patient cohort., Genetic variants in ACTB and ACTG1 have been associated with Baraitser-Winter Cerebrofrontofacial syndrome. Here, the authors report of a syndromic thrombocytopenia caused by variants in ACTB exons 5 or 6 that compromise the organization and coupling of the cytoskeleton, leading to impaired platelet maturation.

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, eLife digest Our muscles contain large numbers of ‘motor proteins’ called myosins. To contract a muscle, many myosin molecules expend energy to ‘walk’ along a filament made from another molecule, called actin, and generate a pulling force. Like other proteins, myosins must fold into the correct shape to work, but high temperatures or other types of stress can disrupt their ability to adopt or maintain the correct shape. Misfolding of myosins, for example, can result in muscular diseases, including those that affect the heart; so there is an ongoing effort to find compounds that can stabilize protein folding and treat these diseases. The small molecule EMD 57033 was discovered over 20 years ago, and its ability to increase the strength of muscle contractions suggested that it could be used to treat chronic heart failure, but the risk of side effects limited its clinical use. The effectiveness of other compounds that improve cardiac muscle function is still routinely compared to EMD 57033, however the exact mechanism responsible for its effect on muscle tissue remained unknown. Now Radke, Taft et al. have identified the part of the myosin protein that EMD 57033 binds to, and shown how this activates muscle contraction. The experiments also, unexpectedly, revealed that EMD 57033 is able to convert misfolded myosin back into the fully functional form. By revealing this refolding effect, the findings of Radtke, Taft et al. suggest that similar small molecules could be used as drugs for the treatment of protein misfolding diseases, muscular diseases, and heart failure. DOI: http://dx.doi.org/10.7554/eLife.01603.002

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.

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.

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.

Molecular motors of the myosin superfamily share a generic motor domain region. They bind actin in an ATP-sensitive manner, exhibit actin-activated ATPase activity, and generate force and movement in this interaction. Many myosins, such as the members of class-18 have not been characterized in-depth. Class-18 myosins form heavy chain dimers and contain protein interaction domains outside the motor domain. Changes of otherwise highly conserved active site residues have raised the question whether myosin-18 can productively interact with ATP in the same way as other myosins. A recently described interaction of mammalian myosin-18A with the Golgi-associated phosphoprotein GOLPH3 was proposed to link the Golgi apparatus to the actin-based cytoskeleton. Here, we characterized human myosin-18A function and its interaction with GOLPH3 in vitro. We found that the interaction of the myosin-18A motor domain with actin filaments appears to be unusual, as the ADP state displays the highest actin-affinity. We performed negative-stain electron microscopy with myosin-18A motor domain decorated F-actin filaments in different nucleotide states and observed the most homogenous and continuous decoration in the ADP-state. The isolated N-terminal subdomain contains a region rich in lysine and glutamate (KE) and a PDZ module. We show that the KE-rich region displays ATP-independent binding to F-actin, whereas the PDZ module mediates the binding of myosin-18A to GOLPH3 and thereby to the Golgi apparatus. We propose that human myosin-18A connects the actin cytoskeleton with the surfaces of organelles such as the Golgi apparatus and that it can target interacting proteins or complexes to the actin cytoskeleton via its PDZ module.

Class-18 myosins challenge our established view about myosins acting as molecular motors. No member of this class appears to have a significant ATPase activity, which is a prerequisite for motor activity. Humans express two myosin-18 isoforms, myosin-18A and myosin-18B. Whereas recent studies on myosin-18A shed some light on its cellular and biochemical mode of action, the molecular function of myosin-18B remains poorly understood. Class-18 myosins contain protein interaction domains outside their generic motor domain. In the case of myosin-18B this includes a large, N-terminal extension that shows no similarity to any known protein domain. Here, we show that the human myosin-18B motor domain binds to F-actin with an affinity of 4 µM. The isolated motor domain binds ATP but has no intrinsic ATPase activity. The large N-terminal extension is shown to directly bind to F-actin with an affinity of 7 µM. This interaction is nucleotide-independent but shows strong ionic strength dependence, which is indicative for a charge-mediated actin binding mechanism. We further analyzed the molecular function of the N-terminal extension by means of actin polymerization assays and found that the myosin-18B N-terminus inhibits F-actin assembly in vitro. Myosin-18B has previously been shown to be located in the cytoplasm of undifferentiated myoblasts. At later stages of differentiation it accumulates in myonuclei. Furthermore, it has been shown that cardiomyocytes display a partial sarcomeric pattern of myosin-18B alternating that of α-actinin-2. Based on our data, we propose a role for myosin-18B in the regulation of muscle sarcomere architecture during differentiation and the regulation of the nuclear actin pool.

Nonmuscle myosin-2B (NM-2B) is a class II myosin involved in cellular processes requiring proper tension maintenance in the cytoskeleton such as cell polarity, movement, and division. The expression of MYH10, the gene encoding the NM-2B heavy chain, is tightly regulated in a time and space dependent manner. It has been suggested that the localization and functional competence of NM-2B is influenced by complex formation with specific isoforms of actin and tropomyosin (Tm). In previous work, we have shown that ATP turnover by NM-2B is up to 4-fold increased when skeletal muscle actin is exchanged against cytoplasmic actin isoforms. To study the functional competence of NM-2B in the presence of regulated cytoplasmic actin filaments, we produced human NM-2B, β- and γ-actin, and selected nonmuscle isoforms of tropomyosins in recombinant form. In addition to the functional characterization of isoform-specific NM-2B interactions, we report results obtained by X-ray crystallographic analysis of the NM-2B motor domain. The nucleotide-free motor domain crystallized readily in the monoclinic space group P21 with two molecules in the asymmetric unit. Details of the crystal structure solved to 2.9 A resolution will be described.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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 Mg(2+)-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 Mg(2+)-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.

Nature structural & molecular biology 16(1), 80-88 (2009). doi:10.1038/nsmb.1542, Published by Nature Publishing Group, London [u.a.]

All myosins undergo the same ATP dependent biochemical cycle but with variations in the lifetime of the individual states of the cycle and the fraction of the total cycle time spent in each state. E.g., processive class-5 myosins are high duty ratio motors that stay most of the ATPase cycle time (> 0.5) attached to actin, whereas fast myosins such as muscle myosin-2 and some class-1 myosin members have a low duty ratio (< 0.1). The members of the respective groups are assumed to use different ways to couple conformational changes at the nucleotide binding regions to changes that occur at the actin binding sites. Conserved active-site elements termed switch-1 and switch-2 play a major role in the coupling mechanism; however, whether and to which extent small variations in the sequence of switch-1 and switch-2 affect the duty ratio of a given myosin, thus determining its ability for processive movement, fast contractility or tension bearing remains unresolved. Based on structural considerations and confirmed by mutational analyses, we identified key residues in the nucleotide binding pocket that are responsible for making ADP dissociation kinetics dependent on the concentration of free magnesium ions. The exchange of a single amino acid in switch-2 affected the motor properties of all myosins tested, but also transformed low duty ratio motors into high duty ratio and vice versa. In addition, x-ray structural analyses and molecular modeling allowed us to relate the observed changes to altered coupling between the active sites and actin binding regions. These results, together with our cell biological studies demonstrating for the first time that magnesium ions have a regulatory role on motor protein function in vivo reveal that, switch-2 can act as magnesium sensor critically determining the mechanochemical properties of myosins.

Myosins play a crucial role for a wide range of physiological functions. The dysfunction or absence of myosin motors has been linked to diseases such as muscle wasting, heart failure, blood disorders, severe forms of senso-neuronal degeneration and cancer. Therefore, the development of selective and potent small-molecule myosin effectors holds the promise to open routes for the treatment of a wide range of disabling diseases. In this study, we show that the thiadiazinone derivative EMD57033 acts as a pharmacological chaperone for several members of the myosin family. Our results show that EMD57033 stimulates the actin-activated ATPase activity and motor activity of myosins from classes 2,5 and 6 up to 3-fold. The most potent interaction was observed with cardiac β-myosin. EMD57033 lowers the apparent second binding constant for F-actin binding in the presence of ATP from 31 μM (+/- 6 μM) to 4 μM (+/- 1 μM). The KM value for ATP remains unaffected by the drug. Furthermore, addition of 10 μM EMD57033 rescues the motor function of “aged” cardiac β-myosin preparations that display strongly reduced activity in the in vitro motility assay prior to addition of the drug. Structural studies indicate that EMD57033 binds in a pocket that is located between the N-terminal SH3-like subdomain and the motor domain-lever arm junction. The participation of the N-terminal SH3-like subdomain in EMD57033 binding is supported by the failure of class-1 myosins, which lack this domain, to productively interact with EMD57033.