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The effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1, and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin.

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.

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.

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.

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

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.