Structural interpretation of the two-site binding of troponin on the muscle thin filament

Conditions are described for the quantitative removal of amino acid residues 274 to 284 from rabbit muscle α-tropomyosin with carboxypeptidase A. The product, non-polymerizable tropomyosin, has a much reduced affinity for the tropomyosinbinding fragment CB1 (residues 1 to 151) of troponin-T. Iodination of α-tropomyosin and non-polymerizable tropomyosin by ¹²⁵I and lactoperoxidase was carried out in the presence and absence of CB1. Following tryptic digestion and peptide mapping, the radioactivities of the labeled tyrosine peptides were compared. In the presence of CB1, tyrosine residues 261 and 267 were iodinated only to the extent of 30 to 40% as compared with the same tyrosine residues in the absence of CB1, All other tyrosine residues (60, 162, 214 and 221) were iodinated to a similar level in the absence or presence of CB1. With non-polymerizable tropomyosin, the presence of CB1 had a much reduced effect on the level of labeling of the tyrosine residues. We conclude that the highly helical region of troponin-T (residues 71 to 151) binds close to or at the COOH-terminal end of the tropomyosin molecule. Taken together with other considerations and recent observations, the results can be interpreted in terms of the two-site model for troponin attachment to the thin filament. A calcium-insensitive site would involve interaction of the highly helical CB2 region of troponin-T (residues 71 to 151) and the COOH-terminal region of tropomyosin (residues 258 to 284) and perhaps the NH₂-terminal overlap region (residues 1 to 9). A calcium-sensitive site would involve the interaction of troponin-T in the neighborhood of cysteine 190 of tropomyosin in F-actin-tropomyosin assemblies both directly and indirectly through the association of its COOH and NH₂-terminal regions with the troponin-I and C components.     

Diversity in smooth muscle thin filament composition

Two classes of smooth muscle thin filament can be identified and separated based on their interaction with antibodies specific either to filamin or to caldesmon. One type is composed of actin, tropomyosin and filamin and the other of actin, tropomyosin and caldesmon.     

Structural changes in the muscle thin filament during contractions caused by single and double electrical pulses

In order to investigate the structural changes of the myofilaments involved in the phenomenon of summation in skeletal muscle contraction, we studied small-angle x-ray intensity changes during twitches of frog skeletal muscle elicited by either a single or a double stimulus at 16 °C. The separation of the pulses in the double-pulse stimulation was either 15 or 30 ms. The peak tension was more than doubled by the second stimulus. The equatorial (1,0) intensity, which decreased upon the first stimulus, further decreased with the second stimulus, indicating that more cross-bridges are formed. The meridional reflections from troponin at 1/38.5 and 1/19.2 nm^− 1 were affected only slightly by the second stimulus, showing that attachment of a small number of myosin heads to actin can make a cooperative structural change. In overstretched muscle, the intensity increase of the troponin reflection in response to the second stimulus was smaller than that to the first stimulus. These results show that the summation is not due to an increased Ca binding to troponin and further suggest a highly cooperative nature of the structural changes in the thin filament that are related to the regulation of contraction.     

Structural changes in the thin filament during activation studied by X-ray diffraction of highly stretched skeletal muscle

The actin layer-lines were recorded from a frog semitendinosus muscle stretched to a sarcomere length greater than 4.4 microM. On activation of the muscle, the equator, the second layer-line at 1/18 nm-1 and the 5.9 nm layer-line increased in integrated intensity. On the other hand, the integrated intensity of the first layer-line at 1/36 nm-1 decreased markedly on activation. This decrease was not fully attributable to shifts of tropomyosin strands and therefore suggested a structural change in the actin subunit. The decrease may account for the apparent lack of an intensity increase of this layer-line on activation at normal muscle lengths where attachment of myosin heads to actin increases the intensities of other layer-lines.     

Unusual thick and thin filament packing in a crustacean muscle

The proximal accessory flexor (PAF) of the myochordotonal organ (MCO) in the meropodite of crayfish walking legs contains two populations of muscle fibers which are distinguishable by their diameters. The large accessory (LA) fibers are 40-80 micrometer in diam and are similar in ultrastructure to other slow crustacean fibers. The small accessory (SA) fibers are 1-12 micrometer in diam and have a unique myofilament distribution at normal body lengths. There is extensive double overlap of thin filaments at these lengths, and some of them form bundles that may extend the length of the sarcomere. In the middle of the sarcomeres, thick and thin filaments are totally segregated from each other. When the fibers are stretched to lengths beyond double overlap length, the myofilament patterns are conventional. The segregated pattern is reestablished when stretched fibers are allowed to shorten passively. The length-tension relationship of the SA fibers is described by a linear ascending branch, a plateau, and a linear descending branch. The ascending branch encompasses normal body lengths from slack length (Ls) with maximum double overlap to the length at which double overlap ceases (1.8 X Ls). The descending phase is comparable to that of other skeletal muscles. That is, tension decreases in proportion with the reduction in thick-thin filament interdigitation (2 X Ls to 3 X Ls).     

Role of actin C-terminus in regulation of striated muscle thin filament

In striated muscle, regulation of actin-myosin interactions depends on a series of conformational changes within the thin filament that result in a shifting of the tropomyosin-troponin complex between distinct locations on actin. The major factors activating the filament are Ca²⁺ and strongly bound myosin heads. Many lines of evidence also point to an active role of actin in the regulation. Involvement of the actin C-terminus in binding of tropomyosin-troponin in different activation states and the regulation of actin-myosin interactions were examined using actin modified by proteolytic removal of three C-terminal amino acids. Actin C-terminal modification has no effect on the binding of tropomyosin or tropomyosin-troponin + Ca²⁺, but it reduces tropomyosin-troponin affinity in the absence of Ca²⁺. In contrast, myosin S1 induces binding of tropomyosin to truncated actin more readily than to native actin. The rate of actin-activated myosin S1 ATPase activity is reduced by actin truncation both in the absence and presence of tropomyosin. The Ca²⁺-dependent regulation of the ATPase activity is preserved. Without Ca²⁺ the ATPase activity is fully inhibited, but in the presence of Ca²⁺ the activation does not reach the level observed for native actin. The results suggest that through long-range allosteric interactions the actin C-terminus participates in the thin filament regulation.     

Characterization of three regulatory states of the striated muscle thin filament

The troponin-tropomyosin-linked regulation of striated muscle contraction occurs through allosteric control by both Ca(2+) and myosin. The thin filament fluctuates between two extreme states: the inactive "off" state and the active "on" state. Intermediate states have been proposed from structural studies and transient kinetic measurements. However, in contrast to the well-characterised, on and off states, the mechanochemical properties of the intermediate states are much less well understood because of the instability of those states. In the present study, we have characterized a myosin-induced intermediate that is stabilized by cross-linking myosin motor domains (S1) to actin filaments (with a maximum of one S1 molecule for 50 actin monomers). A single S1 molecule is known to interact with two adjacent actin monomers. A detailed analysis revealed that thin filaments containing S1 molecules cross-linked to just one actin monomer (actin(1)-S1 complexes) are regulated with a 79% inhibition of the ATPase in the absence of Ca(2+). In contrast, filaments containing S1 molecules cross-linked at two positions, to two adjacent actin monomers (actin(2)-S1 complexes) totally lose their regulation in a highly cooperative manner. This loss of regulation was due both to an enhancement of the ATPase activity without calcium and an inhibition of the ATPase with calcium. Filaments containing actin(2)-S1 complexes, with significant ATPase activity in the absence of calcium (about 50%), did not move on a myosin-coated surface unless calcium was present. This partial uncoupling between the ATPase activity and in vitro motility in the absence of calcium demonstrates that the mechanical steps require actin-myosin contacts, which take place only in the on state and not in the off or intermediate states. These data provide new insights concerning the difference in cooperativity of Ca(2+) regulation that exists between the biochemical and mechanical cycles of the actin-myosin motor.     

Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins

Caldesmon binds equally to both gizzard actin and actin containing stoichiometric amounts of bound tropomyosin. The binding of caldesmon to actin inhibits the actin-activation of the Mg-ATPase activity of phosphorylated myosin only when the actin contains bound tropomyosin. The reversal of this inhibition requires Ca2+-calmodulin; but it occurs without complete release of bound caldesmon. Although phosphorylation of the caldesmon occurs during the ATPase assay, a direct correlation between caldesmon phosphorylation and the release of the inhibited actomyosin ATPase is not consistently observed.     

Calcium regulation of skeletal muscle thin filament motility in vitro.

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.     

Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction

Recent developments in the field of myofibrillar proteins will be reviewed. Consideration will be given to the proteins that participate in the contractile process itself as well as to those involved in Ca-dependent regulation of striated (skeletal and cardiac) and smooth muscle. The relation of protein structure to function will be emphasized and the relation of various physiologically and histochemically defined fiber types to the proteins found in them will be discussed.     

Reconstitution of the muscle thin filament from recombinant troponin components and the native thin filaments

We have developed a technique by which muscle thin filaments are reconstituted from the recombinant troponin components and the native thin filaments. By this technique, the reconstituted troponin complex is exchanged into the native thin filaments in the presence of 20% glycerol and 0.3 M KCl at pH 6.2. More than 90% of endogenous troponin complex was replaced with the recombinant troponin complex. Structural integrity and Ca²⁺ sensitivity of the reconstituted thin filament prepared by this technique was confirmed by X-ray fiber diffraction measurements and the thin filament-activated myosin subfragment 1 ATPase measurements, respectively.     

Mode of caldesmon binding to smooth muscle thin filament: possible projection of the amino-terminal of caldesmon from native thin filament.

The structure of smooth muscle thin filament was examined by various electron microscopy techniques, with special attention to the mode of caldesmon binding. Chemical cross-linking was positively used to avoid the dissociation of accessory proteins upon dilution. Caldesmon in reconstituted thin filament was observed as fine filamentous projections from thin filament. Native thin filament isolated from smooth muscle showed similarly numerous fine whisker-like projections by all the techniques employed here. Antibody against the amino-terminus of caldesmon labeled the end of such projections indicating the possibility that the amino-terminal myosin binding moiety might stick out from the shaft of the thin filament. Such whiskers are often projected out as a cluster to the same side of native thin filament. Further, we could visualize the assembly of dephosphorylated heavy meromyosin (HMM) with native or reconstituted thin filament forming "nonproductive" complex in the presence of ATP. The association of HMM to the shaft of thin filament was through subfragment-2 moiety, in accordance with biochemical studies. Some HMM particles bound closer to the thin filament shaft, possibly suggesting the presence of the second myosin-binding site on caldesmon. Occasionally two kinds of HMM association as such coexisted at a single site on this filament in tandem. Thus, we constructed a structural model of thin filament. The proposed molecular arrangement is not only compatible with all the biochemical results but also provides additional support for our recent findings (E. Katayoma, G. C. Scott-Woo, and M. Ikebe (1995) J. Biol. Chem. 270, 3919-3925) regarding the capability of caldesmon to induce dephosphorylated myosin filament, which explains the existence of thick filaments in relaxed smooth muscle cells.     

Binding of phosphorylase a and b to skeletal muscle thin filament proteins

Phosphorylase plays an important role in energy generation during muscle contraction. We have demonstrated that purified rabbit skeletal muscle phosphorylase a and phosphorylase b bind to rabbit muscle F-actin, F-actin-tropomyosin, F-actin-tropomyosin-troponin, and myofibrils. Neither phosphorylase a nor phosphorylase b binds to myosin. Phosphorylase a and b bind to F-actin with S0.5 values of 1.5 X 10(-6) and 2.1 X 10(-6) M, respectively. At saturation, 0.035 mol of phosphorylase a and b is bound for every seven G-actin monomers in the F-actin polymer. Using the F-actin-tropomyosin-troponin complex as opposed to F-actin as a binding target, there are five- and threefold increases in the maximal binding capacity for phosphorylase a and phosphorylase b, respectively, without a significant change in the S0.5 value for either form of the enzyme. A similar stoichiometry and affinity of phosphorylase binding are observed when myofibrils are used as the binding target. Ca2+ ions and AMP increase the maximal binding capacity for phosphorylase a to myofibrils while ATP decreases the Bmax. Our study suggests that in skeletal muscle, phosphorylase a and phosphorylase b may interact with the thin filament, and that this binding to thin filament proteins may be controlled by changes in sarcoplasmic concentration of Ca2+ and ligands of phosphorylase during muscle contraction.     

The thin filament of vertebrate skeletal muscle co-operatively activates as a unit

We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca2+ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca2+ binding protein responsible for the steep slope of the pCa/tension relation.     

Structural dynamics of C-domain of cardiac troponin I protein in reconstituted thin filament

The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca(2+) binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca(2+)-bound to the Ca(2+)-free dynamic states were significantly faster (53.2-116.8 s(-1)) than that of the IR (14.1 s(-1)). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca(2+) dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca(2+) signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.     

Actin dynamics at pointed ends regulates thin filament length in striated muscle

Regulation of actin dynamics at filament ends determines the organization and turnover of actin cytoskeletal structures. In striated muscle, it is believed that tight capping of the fast-growing (barbed) ends by CapZ and of the slow-growing (pointed) ends by tropomodulin (Tmod) stabilizes the uniform lengths of actin (thin) filaments in myofibrils. Here we demonstrate for the first time that both CapZ and Tmod are dynamic on the basis of the rapid incorporation of microinjected rhodamine-labelled actin (rho-actin) at both barbed and pointed ends and from the photobleaching of green fluorescent protein (GFP)-labelled Tmod. Unexpectedly, the inhibition of actin dynamics at pointed ends by GFP-Tmod overexpression results in shorter thin filaments, whereas the inhibition of actin dynamics at barbed ends by cytochalasin D has no effect on length. These data demonstrate that the actin filaments in myofibrils are relatively dynamic despite the presence of capping proteins, and that regulated actin assembly at pointed ends determines the length of thin filaments.     

Number of anti-troponin striations along the thin filament of chick embryonic breast muscle

Anti-troponin formed 25 to 29 striations with a period of 38 nm along the whole length of thin filaments in chick embryonic breast muscle, in contrast with the uniform formation of 24 striations in adult muscle. This indicates that the thin filament in embryonic breast muscle is longer than that in the adult muscle.