A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146–174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca²⁺. Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146–174 on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41–69, 83–111, 216–244, and 252–279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217–236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca²⁺ than with it. Ca²⁺-induced changes on the actin–Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.     

Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.     

The role of tropomyosin domains in cooperative activation of the actin-myosin interaction

To establish α-tropomyosin (Tm)'s structure–function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm–actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca²⁺. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm–actin interaction cooperatively affects actin to result in generation of greater force and velocity.     

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.     

Folic acid supplementation dysregulates gene expression in lymphoblastoid cells--implications in nutrition

For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ?four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics.     

Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments.     

FBN1 isoform expression varies in a tissue and development-specific fashion

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54 and 55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8–44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39 ± 3 (%, mean ± SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19 ± 2 (%, mean ± SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9–13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.     

Smcy transgene does not rescue spermatogenesis in sex-reversed mice

In mouse, the Sxr^b deletion interval (delta Sxr^b) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1–2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr^b interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr^b/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy. Received: 16 June 2000 / Accepted: 25 September 2000     

Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Conserved Asp-137 imparts flexibility to tropomyosin and affects function

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.     

Substrate specificity of diacylglycerol kinase-epsilon and the phosphatidylinositol cycle

We show that diacylglycerol kinase-ε (DGKε) has less preference for the acyl chain at the sn-1 position of diacylglycerol (DAG) than the one at the sn-2 position. Although DGKε discriminates between 1-stearoyl-2-arachidonoyl-DAG and 1-palmitoyl-2-arachidonoyl-DAG, it has similar substrate preference for 1-stearoyl-2-arachidonoyl-DAG and 1,2-diarachidonoyl-DAG. We suggest that in addition to binding to the enzyme, the acyl chain at the sn-1 position may contribute to the depth of insertion of the DAG into the membrane. Thus, the DAG intermediate of the PI-cycle, 1-stearoyl-2-arachidonoyl-DAG, is not the only DAG that is a good substrate for DGKε, the DGK isoform involved in PI-cycling.     

Regulation of β-tubulin function and expression in Drosophila spermatogenesis

In this study we examined two aspects of β-tubulin function in Drosophila spermatogenesis: 1) β-tubulin structural requirements for assembly of different categories of microtubules and 2) regulatory requirements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific β2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, β3, cannot support spermatogenesis, whereas a carboxyl-truncated form of β2, β2ΔC, can at least to some extent provide all of β2′s normal functions, save one: β2ΔC cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whether β2 carboxyl sequences can rescue the functional failure of the β3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, β3β2C, in which β3 sequences in the carboxyl region are replaced with those of β2. Unlike either β3 or β2ΔC, β3β2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the β2 carboxyl sequences mediate morphogenesis of the axoneme doublet tubule complex, including accessory microtubule assembly and attachment of spokes and linkers. However, our data also reveal aspects of β2-specific function that require structural features other than the primary sequence of the isotype-defining variable regions, the C terminus and the internal variable region. Tests of fecundity in males that co-express Δ2 and the chimeric Δ3Δ2C protein showed that in Drosophila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubule function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in the male germ cells. We found that both Δ2-tubulin mRNA accumulation and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3′ noncoding sequences in the Δ2 gene. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observed in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of × chromosome expression in Drosophila spermatogenesis. © 1995 Wiley-Liss, Inc.     

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.     

Crystal Structures of Human HMG-CoA Synthase Isoforms Provide Insights into Inherited Ketogenesis Disorders and Inhibitor Design

3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.     

Cryo-EM Structures of the Actin:Tropomyosin Filament Reveal the Mechanism for the Transition from C- to M-State

Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of Tm along the actin (Ac):Tm:troponin (Tn) thin filament to block or expose myosin binding sites on Ac. In striated muscle, including involuntary cardiac muscle, Tm regulates muscle contraction by coupling Ca^2 + binding to Tn with myosin binding to the thin filament. In smooth muscle, the switch is the posttranslational modification of the myosin. Depending on the activation state of Tn and the binding state of myosin, Tm can occupy the blocked, closed, or open position on Ac. Using native cryogenic 3DEM (three-dimensional electron microscopy), we have directly resolved and visualized cardiac and gizzard muscle Tm on filamentous Ac in the position that corresponds to the closed state. From the 8-Å-resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm, we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed-state position.     

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.