Structural Interactions Responsible for the Assembly of the Troponin Complex on the Muscle Thin Filament (original) (raw)
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The troponin complex and regulation of muscle contraction
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1995
In a wide variety of cellular settings, from organelle transport to muscle contraction, Ca2+ binding to members of the EF hand family of proteins controls the interaction between actin and different myosins that are responsible for generating movement. In vertebrate skeletal and cardiac muscle the Ca(2+)-binding protein troponin C (TnC) is one subunit of the ternary troponin complex which, through its association with actin and tropomyosin on the thin filament, inhibits the actomyosin interaction at submicromolar Ca2+ concentrations and stimulates the interaction at micromolar Ca2+ concentrations. Because TnC does not interact directly with actin or tropomyosin, the Ca(2+)-binding signal must be transmitted to the thin filament via the other two troponin subunits: troponin I (TnI), the inhibitory subunit, and troponin T (TnT), the tropomyosin-binding subunit. Thus, the troponin complex is a Ca(2+)-sensitive molecular switch and the structures of and interactions between its componen...
Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin
Journal of Molecular Biology, 2009
Abbreviations used: TnI, the inhibitory subunit of troponin; cTerm-TnI, the C-terminal 80 amino acid domain of TnI that links to actin at low Ca 2+ ; TnC, the Ca 2+ -sensor of troponin that releases inhibition; TnT, the element linking troponin to tropomyosin; B-state, the blocked state; C-state, the closed state; M-state, the open state.
Journal of Biological Chemistry, 2000
The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(⌬96 -116)), 2) the COOH-terminal domain (TnI-(⌬105-115)), and 3) the NH 2 -terminal domain (TnI-(⌬95-106)). Measurements of Ca 2؉ -regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI⅐TnC complex restored the Ca 2؉ sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(⌬96 -116)⅐TnC and TnI-(⌬105-115)⅐TnC. The TnI-(⌬95-106)⅐TnC mutant retained ϳ55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca 2؉ sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca 2؉ , and two of them (TnI-(⌬96 -116) and TnI-(⌬105-115)) gave significant activation in the absence of Ca 2؉ . These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH 2 -terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca 2؉ -sensitive molecular switch during muscle contraction.
Structural dynamics of troponin during activation of skeletal muscle
Proceedings of the National Academy of Sciences, 2014
Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s −1 ) matches the rate of Ca 2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s −1 ) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s −1 ) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions. muscle regulation | excitation-contraction coupling | muscle signaling C ontraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca 2+ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2-4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.
Structural Basis for the Regulation of Muscle Contraction by Troponin and Tropomyosin
Journal of Molecular Biology, 2008
The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca 2+ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosincrossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca 2+ -binding to TnC presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI with tropomyosin and actin that cause tropomyosin movement are unknown and thus the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI and then 3D-EM was used to resolve where troponin is anchored on actin-tropomyosin. EM-reconstruction showed how TnI-binding to both actin and tropomyosin at low-Ca 2+ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated stericinterference of actin-myosin interaction that regulates muscle contraction.
Journal of Molecular Biology, 2009
The Ca 2+ -dependent interaction of troponin I (TnI) with actin•tropomyosin (Actin•Tm) in the muscle thin filament is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca 2+ , TnI interacts with Tm as well as actin in the reconstituted muscle thin filament, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone (BPmal) at position 146 or 174 (Tm146* or Tm174*, respectively), reconstituted with actin and troponin (composed of TnI; troponin T, TnT; and troponin C, TnC) or with actin and TnI. After near uv-irradiation, SDS gels of the Tm*146containing thin filament showed 3 new high molecular weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-TnC and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry and Western blots. While Tm*146-TnI was produced only in the absence of Ca 2+ , the production of the other crosslinked species did not show a Ca 2+ dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI of MW 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and MALDI-TOF. This was shown, using absorption and fluorescence spectroscopy, to be the BPmal-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data showing that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca 2+ support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin, and will help efforts to localize Tn in the actin•Tm muscle thin filament.
Biochemistry, 2006
Rabbit skeletal muscle R-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca 2+ -sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg 2+ -ATPase rate regardless of whether Ca 2+ and Tn were present. On the other hand, mTm134-147 retained the normal Ca 2+ -sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actininduced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.
A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation
Journal of Biological Chemistry, 2002
In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI-TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT 1 tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT 1 producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C-to M-state equilibrium, K T , for TnT 1 . This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.
The role of tropomyosin-troponin in the regulation of skeletal muscle contraction
Journal of Muscle Research and Cell Motility, 1986
Steric blocking of actin-myosin interaction by tropomyosin has been a working hypothesis in the study of the regulation of skeletal muscle contraction, yet the simple movement of actin-associated tropomyosin from a myosin-blocking position (relaxation) to a nonblocking position (contraction) cannot adequately account for all of the biophysical and biochemical observations which have been made to date. Ambiguous assignment of tropomyosin positions on actin during contraction, due in part to the limited resolution of reconstruction techniques, may also hint at a real lack of clearcut 'on' and 'off' positioning of tropomyosin and tropomyosin-troponin complex. Recent biochemical evidence suggests processes relatively independent of tropomyosin-troponin may have a governing effect on contraction, involving kinetic constraints on actin-myosin interaction influenced by the binding of ATP and the intermediates of ATP hydrolysis. Based on our current understanding put forth in this review, it is clear that regulatory interactions in muscle contraction do not consist solely of steric effects but involve kinetic factors as well. Where the latter are being defined in systems reconstituted from purified proteins and their fragments, the steric components of regulation are most clearly observed in studies of structurally more intact physiologic systems (e.g. intact or skinned whole muscle fibres). The fine detail of the processes and their interplay remains an intriguing question. Likewise, the precise physical relationship of myosin with actin in the crossbridge cycle continues to elude definition. Refinement of several methodologies (X-ray crystallography, three-dimensional reconstruction, time-resolved X-ray diffraction) will increase the potential for detailing the molecular basis of the regulation of muscle contraction.