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Biophysical Journal Sep 2018Often considered an archetypal dimeric coiled coil, tropomyosin nonetheless exhibits distinctive "noncanonical" core residues located at the hydrophobic interface...
Often considered an archetypal dimeric coiled coil, tropomyosin nonetheless exhibits distinctive "noncanonical" core residues located at the hydrophobic interface between its component α-helices. Notably, a charged aspartate, D137, takes the place of nonpolar residues otherwise present. Much speculation has been offered to rationalize potential local coiled-coil instability stemming from D137 and its effect on regulatory transitions of tropomyosin over actin filaments. Although experimental approaches such as electron cryomicroscopy reconstruction are optimal for defining average tropomyosin positions on actin filaments, to date, these methods have not captured the dynamics of tropomyosin residues clustered around position 137 or elsewhere. In contrast, computational biochemistry, involving molecular dynamics simulation, is a compelling choice to extend the understanding of local and global tropomyosin behavior on actin filaments at high resolution. Here, we report on molecular dynamics simulation of actin-free and actin-associated tropomyosin, showing noncanonical residue D137 as a locus for tropomyosin twist variation, with marked effects on actin-tropomyosin interactions. We conclude that D137-sponsored coiled-coil twisting is likely to optimize electrostatic side-chain contacts between tropomyosin and actin on the assembled thin filament, while offsetting disparities between tropomyosin pseudorepeat and actin subunit periodicities. We find that D137 has only minor local effects on tropomyosin coiled-coil flexibility, (i.e., on its flexural mobility). Indeed, D137-associated overtwisting may actually augment tropomyosin stiffness on actin filaments. Accordingly, such twisting-induced stiffness of tropomyosin is expected to enhance cooperative regulatory translocation of the tropomyosin cable over actin.
Topics: Actins; Amino Acid Sequence; Molecular Dynamics Simulation; Protein Binding; Protein Conformation, alpha-Helical; Tropomyosin
PubMed: 30195938
DOI: 10.1016/j.bpj.2018.08.017 -
BMC Biology May 2020Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits,...
BACKGROUND
Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.
RESULTS
Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.
CONCLUSIONS
Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.
Topics: Blood Platelets; CRISPR-Cas Systems; Genome-Wide Association Study; Hematopoiesis; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Tropomyosin
PubMed: 32408895
DOI: 10.1186/s12915-020-00783-7 -
Journal of Muscle Research and Cell... Aug 2013Tropomyosin (Tm) is the key regulatory component of the thin-filament and plays a central role in the cardiac muscle's cooperative activation mechanism. Many mutations... (Review)
Review
Tropomyosin (Tm) is the key regulatory component of the thin-filament and plays a central role in the cardiac muscle's cooperative activation mechanism. Many mutations of cardiac Tm are related to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC). Using the thin-filament extraction/reconstitution technique, we are able to incorporate various Tm mutants and protein isoforms into a muscle fiber environment to study their roles in Ca(2+) regulation, cross-bridge kinetics, and force generation. The thin-filament reconstitution technique poses several advantages compared to other in vitro and in vivo methods: (1) Tm mutants and isoforms are placed into the real muscle fiber environment to exhibit their effect on a level much higher than simple protein complexes; (2) only the primary and immediate effects of Tm mutants are studied in the thin-filament reconstituted myocardium; (3) lethal mutants of Tm can be studied without causing a problem; and (4) inexpensive. In transgenic models, various secondary effects (myocyte disarray, ECM fibrosis, altered protein phosphorylation levels, etc.) also affect the performance of the myocardium, making it very difficult to isolate the primary effect of the mutation. Our studies on Tm have demonstrated that: (1) Tm positively enhances the hydrophobic interaction between actin and myosin in the "closed state", which in turn enhances the isometric tension; (2) Tm's seven periodical repeats carry distinct functions, with the 3rd period being essential for the tension enhancement; (3) Tm mutants lead to HCM by impairing the relaxation on one hand, and lead to DCM by over inhibition of the AM interaction on the other hand. Ca(2+) sensitivity is affected by inorganic phosphate, ionic strength, and phosphorylation of constituent proteins; hence it may not be the primary cause of the pathogenesis. Here, we review our current knowledge regarding Tm's effect on the actomyosin interaction and the early molecular pathogenesis of Tm mutation related to HCM, DCM, and LVNC.
Topics: Animals; Cardiomyopathy, Dilated; Cardiomyopathy, Hypertrophic; Heart; Heart Diseases; Humans; Myocardium; Tropomyosin
PubMed: 23700264
DOI: 10.1007/s10974-013-9343-z -
Biophysical Journal Jun 2019The initial binding of tropomyosin onto actin filaments and then its polymerization into continuous cables on the filament surface must be precisely tuned to overall...
The initial binding of tropomyosin onto actin filaments and then its polymerization into continuous cables on the filament surface must be precisely tuned to overall thin-filament structure, function, and performance. Low-affinity interaction of tropomyosin with actin has to be sufficiently strong to localize the tropomyosin on actin, yet not so tight that regulatory movement on filaments is curtailed. Likewise, head-to-tail association of tropomyosin molecules must be favorable enough to promote tropomyosin cable formation but not so tenacious that polymerization precedes filament binding. Arguably, little molecular detail on early tropomyosin binding steps has been revealed since Wegner's seminal studies on filament assembly almost 40 years ago. Thus, interpretation of mutation-based actin-tropomyosin binding anomalies leading to cardiomyopathies cannot be described fully. In vitro, tropomyosin binding is masked by explosive tropomyosin polymerization once cable formation is initiated on actin filaments. In contrast, in silico analysis, characterizing molecular dynamics simulations of single wild-type and mutant tropomyosin molecules on F-actin, is not complicated by tropomyosin polymerization at all. In fact, molecular dynamics performed here demonstrates that a midpiece tropomyosin domain is essential for normal actin-tropomyosin interaction and that this interaction is strictly conserved in a number of tropomyosin mutant species. Elsewhere along these mutant molecules, twisting and bending corrupts the tropomyosin superhelices as they "lose their grip" on F-actin. We propose that residual interactions displayed by these mutant tropomyosin structures with actin mimic ones that occur in early stages of thin-filament generation, as if the mutants are recapitulating the assembly process but in reverse. We conclude therefore that an initial binding step in tropomyosin assembly onto actin involves interaction of the essential centrally located domain.
Topics: Actins; Amino Acid Sequence; Molecular Dynamics Simulation; Mutation; Protein Binding; Protein Structure, Secondary; Tropomyosin
PubMed: 31130236
DOI: 10.1016/j.bpj.2019.05.009 -
Biophysical Journal Feb 2011Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules...
Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin filaments, free of constraints imposed by troponin or other actin-binding proteins, cannot be formulated, and thus optimal interfacial contacts between actin and tropomyosin remain unknown. Here, a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions, and pseudo-rotations of tropomyosin on F-actin was performed. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic residues on the successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into electron microscopy reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the seven broad and narrow motifs that typify tropomyosin's twisting superhelical coiled-coil to the wide and tapering tropomyosin densities seen in surface views of F-actin-tropomyosin reconstructions. The functional implications of the F-actin-tropomyosin models determined in this work are discussed.
Topics: Actins; Amino Acids; Animals; Computer Simulation; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Microscopy, Electron; Models, Molecular; Protein Binding; Rabbits; Reproducibility of Results; Static Electricity; Tropomyosin
PubMed: 21320445
DOI: 10.1016/j.bpj.2010.12.3697 -
Journal of Structural Biology May 2010Complementarity between the tropomyosin supercoil and the helical contour of actin-filaments is required for the binding interaction of actin and tropomyosin (Li et al.,...
Complementarity between the tropomyosin supercoil and the helical contour of actin-filaments is required for the binding interaction of actin and tropomyosin (Li et al., 2010). Clusters of small alanine residues in place of canonical leucines along coiled-coil tropomyosin may be responsible for pre-shaping tropomyosin and promoting conformational complementarity to F-actin. A longitudinal displacement between the two chains of the tropomyosin coiled-coil induced by the alanine clusters could produce localized bending or limited flexibility along tropomyosin needed to shape tropomyosin (Brown and Cohen, 2005). To evaluate the influence of alanine clusters on tropomyosin curvature, we calculated the longitudinal displacement between amino acid residues on adjacent chains of the tropomyosin coiled-coil and related this "Z-displacement" to the position of the alanine clusters. Measurements were made on high-resolution crystal structures of tropomyosin fragments and on trajectories from molecular dynamics simulations of full-length alphaalpha-tropomyosin. We found no strict one-for-one spatial correlation between alanine cluster position and the Z-displacement. Neither did we find any direct correspondence between the clusters and the local curvature of tropomyosin. Rather than just causing specific local structural effects, the overall influence of alanine clusters is complex and delocalized, leading to a gradually changing bending pattern along the length of tropomyosin.
Topics: Actins; Alanine; Amino Acid Sequence; Crystallography, X-Ray; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Protein Conformation; Tropomyosin
PubMed: 20026408
DOI: 10.1016/j.jsb.2009.12.017 -
International Archives of Allergy and... 2016The mosquito Aedes aegypti is a potential source of important clinically relevant allergens. However, the allergenicity and cross-reactivity of most of these has not...
BACKGROUND
The mosquito Aedes aegypti is a potential source of important clinically relevant allergens. However, the allergenicity and cross-reactivity of most of these has not been fully described.
METHODS
Natural wild-type mosquito tropomyosin was purified by size exclusion and anionic-exchange chromatography from an A. aegypti extract. Further characterization was accomplished by MALDI-TOF/TOF. Two recombinant variants of tropomyosin were obtained by expression in Escherichia coli. Specific IgE measurement by ELISA and skin tests for mosquito extract were performed in 12 patients with asthma or allergy rhinitis residing on the Caribbean island of Martinique. Cross-reactivity between natural A. aegypti tropomyosin and recombinant tropomyosins from A. aegypti, house dust mite, shrimp and Ascaris lumbricoides was analyzed by ELISA competition.
RESULTS
Four variants of natural tropomyosin were purified. A band of 32 kDa in SDS-PAGE representing 2 tropomyosin variants (Aed a 10.0101 and Aed a 10.0201) reacted with specific IgE of 4 of the 12 (33%) allergic patients and with rabbit polyclonal anti-shrimp tropomyosin. A high degree of cross-reactivity (60-70%) was detected between natural mosquito tropomyosin and Blo t 10, Der p 10 and Lit v 1, and a lower degree with Asc l 3 from A. lumbricoides (<30%). rAed a 10.0101 inhibited IgE binding to natural A. aegypti tropomyosin; however, rAed a 10.0201 showed a low inhibitory capacity.
CONCLUSION
Tropomyosin is a new IgE-binding protein from A. aegypti. Two of the 4 variants identified showed different degree of cross-reactivity with tropomyosins from other arthropods. The potential allergenic role of each variant should be further investigated.
Topics: Adolescent; Adult; Aedes; Allergens; Amino Acid Sequence; Animals; Child; Child, Preschool; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hypersensitivity; Immunoglobulin E; Male; Protein Binding; Proteome; Proteomics; Tropomyosin; Young Adult
PubMed: 27355916
DOI: 10.1159/000447298 -
International Review of Cell and... 2010The actin cytoskeleton is regulated by a variety of actin-binding proteins including those constituting the tropomyosin family. Tropomyosins are coiled-coil dimers that... (Review)
Review
The actin cytoskeleton is regulated by a variety of actin-binding proteins including those constituting the tropomyosin family. Tropomyosins are coiled-coil dimers that bind along the length of actin filaments. In muscles, tropomyosin regulates the interaction of actin-containing thin filaments with myosin-containing thick filaments to allow contraction. In nonmuscle cells where multiple tropomyosin isoforms are expressed, tropomyosins participate in a number of cellular events involving the cytoskeleton. This chapter reviews the current state of the literature regarding tropomyosin structure and function and discusses the evidence that tropomyosins play a role in regulating actin assembly.
Topics: Actins; Amino Acid Sequence; Animals; Calmodulin-Binding Proteins; Cell Membrane; Cytoskeleton; Humans; Molecular Motor Proteins; Molecular Sequence Data; Muscle Contraction; Myosins; Neoplasm Metastasis; Phosphorylation; Protein Isoforms; Tropomyosin
PubMed: 20460184
DOI: 10.1016/S1937-6448(10)81003-2 -
The Journal of Biological Chemistry Nov 1990The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the...
The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin. Binding was examined by fluorescence spectroscopy and affinity chromatography. Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227. The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200. This further focused the primary caldesmon-binding site to residues 201-227. Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon. Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.
Topics: Animals; Binding Sites; Calmodulin-Binding Proteins; Chickens; Chromosome Deletion; Cloning, Molecular; Escherichia coli; Gizzard, Avian; Kinetics; Models, Molecular; Muscle, Smooth; Muscles; Peptide Fragments; Protein Conformation; Restriction Mapping; Spectrometry, Fluorescence; Tropomyosin
PubMed: 2229046
DOI: No ID Found -
Biochemistry Jun 2014The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding...
The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.
Topics: Actins; Adenosine Triphosphatases; Animals; Cell Movement; Humans; Myosin Subfragments; Myosin Type II; Rats; Tropomyosin
PubMed: 24873380
DOI: 10.1021/bi500162z