Cardiomyopathic tropomyosin mutations that increase thin filament Ca2+ sensitivity and tropomyosin N-domain flexibility

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of alpha-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 degrees C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 degrees C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0-1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of thin filament in two settings: Ca2+-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca2+ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca2+ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.     

The regulation of subtilisin-cleaved actin by tropomyosin/troponin

Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin.

The bindings of troponin components to actin and tropomyosin has been studied by cosedimentation with actin and affinity chromatography. It is shown that troponin binds to actin and tropomyosin in the presence and absence of calcium but the binding to actin is sensitive to ionic strength. Troponin-I + C binds to actin-tropomyosin in the absence of calcium but not to actin or tropomyosin alone. Troponin-I binds to actin and the binding is improved in the presence of tropomyosin even though troponin-I does not bind to tropomyosin alone. Troponin-C does not bind to actin or tropomyosin. The results suggest that the binding of troponin by actin is influenced by tropomyosin. A model of regulation by troponin is proposed.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.     

Interaction of tropomyosin with troponin components.

1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.     

Lysine reactivities of tropomyosin complexed with troponin

The relative reactivities of lysine residues of tropomyosin complexed with troponin have been measured in order to locate the binding site of troponin on tropomyosin in a complex between the two native proteins. The lysines were labeled with acetic anhydride using a competitive labeling procedure and the relative reactivities of tropomyosin lysine containing peptides were compared to those from tropomyosin labeled in the absence of troponin (S. E. Hitchcock-DeGregori, S. F. Lewis, and T. M.-T. Chou, (1985) Biochemistry 24, 3305-3314). Analysis of about two-thirds of the lysines indicates that troponin affects the reactivities of lysines along the length of the tropomyosin, indicating long-range effects. The inferred binding site is more extensive than previously reported, about 25 nm, extending from res. 136 to the carboxy-terminus and to res. 30 beyond the end-to-end overlap in the amino-terminal region of the next tropomyosin molecule.     

Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.     

Phosphorylation of an actin.tropomyosin.troponin complex from human skeletal muscle.

A human skeletal actin.tropomyosin.troponin complex was phosphorylated in the presence of [gamma-32 P]ATP, Mg2+, adenosine 3':5'-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 microM cyclic AMP. In the presence of 10(-7) M Ca2+ and protein kinase 0.1 mole of [32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca2+, 5.10(-5) M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [32P]phosphate. Increasing the Ca2+ concentration did not significantly decrease the [32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstitute human skeletal actomyosin made with the [32P] phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca2+.     

Cardiac tropomyosin crystals and their interactions with troponin subunits

Crystals and paracrystals of bovine cardiac tropomyosin and their mixtures with different combinations of troponin subunits were examined in the electron microscope after negative staining. Although the cardiac proteins gave most of the same crystalline and paracrystalline patterns as observed previously with skeletal muscle tropomyosin and troponin, two important differences were noted. Cardiac troponin T was incapable of forming hexagonal networks with either skeletal or cardiac muscle tropomyosins, while skeletal troponin T readily associated in this manner with tropomyosins from either tissue source. Also the characteristic paracrystalline pattern seen with skeletal muscle tropomyosin, troponin T and troponin C only in the presence of calcium was consistently obtained with mixtures of the corresponding cardiac components when calcium was absent.     

The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils

A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin: myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament.     

Purification of tropomyosin, paramyosin, actin, tubulin, troponin and kinases for chemiproteomics and its application to different scientific fields

Background

p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca²⁺-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood.

Methodology/Principal Findings

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca²⁺ wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine.

Conclusion

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.     

The use of actin labelled with N-(1-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin.

A pyrene label attached to Cys-374 of actin has been shown to be a useful probe for monitoring the interaction of actin with myosin subfragments [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38]. We report that the presence of this label decreases the affinity of actin for myosin subfragment 1 by less than a factor of 2. The rate of actin binding is unaffected by the label and the dissociation rate is increased by up to a factor of 2. Both the rate of actin binding to, and the rate of actin dissociation from, heavy meromyosin show two phases when monitored by pyrene fluorescence. Thin filiments reconstituted from pyrene-labelled actin show a 5% increase in pyrene fluorescence on binding Ca2+.     

Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits.

Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.     

Interactions of desensitized actomyosin with tropomyosin, troponin A, troponin B, and polyanions

Troponin B is an inhibitor of the Mg⁺⁺-activated ATPase activity of actomyosin. The inhibitory effect, which is observed, however, depends upon whether tropomyosin is also present. In the absence of tropomyosin the inhibition by troponin B is markedly reduced by increasing the ionic strength from 0.03 to 0.07, but is not affected by calcium up to a concentration of 10^-4 M. Troponin A relieves the inhibition in both the absence and presence of calcium, an effect which is also shown by many polyanions and is illustrated by using RNA. Tropomyosin enhances the inhibitory effect of troponin B and renders it more resistant to increasing ionic strength but it does not make the inhibition calcium-sensitive. However, when troponin A or low concentrations of polyanions are added to troponin B and tropomyosin, the actomyosin ATPase activity becomes calcium-sensitive; i.e., in the presence of tropomyosin, troponin A or polyanions do not relieve the inhibitory action of troponin B in the absence of calcium but only in its presence. In marked contrast to this is the effect of troponin A in the absence of tropomyosin where it neutralizes the effect of troponin B under all conditions. Thus troponin A and the polyanions both confer calcium regulation on the troponin B-tropomyosin system. The similar effects exhibited by troponin A and the polyanions suggest that the addition of net negative charge to troponin B is an important factor in the conferral of calcium sensitivity. It is also clear that tropomyosin is an essential component of the regulatory mechanism.     

Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.