Calcium-Induced Movement of Troponin-I Relative to Actin in Skeletal Muscle Thin Filaments

Tao, Terence; Gong, Bang-Jian; Leavis, Paul C.

doi:10.1126/science.2138356

Cited by 133 publications

(75 citation statements)

References 20 publications

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“…years of intense investigation in many laboratories (22)(23)(24)(25)(26)(27), the molecular details of the Ca 2ϩ -switch mechanism in cardiac troponin remain incompletely understood. To elucidate the switching mechanism, it is necessary to understand intersubunit interactions within the troponin complex as well as the conformational transitions that occur within the individual troponin subunits.…”

Section: Fig 5 Confidence Estimates In the Recovered Mean Distancesmentioning

confidence: 99%

Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

Dong¹,

Robinson²,

Stagg

et al. 2003

Journal of Biological Chemistry

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Cardiac muscle activation is initiated by the binding of Ca 2؉ to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca 2؉ binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150 -165) and the inhibitory region (cTnI-I, residues130 -149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using Fö rster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg 2؉ -saturated) and Ca 2؉ -activated (Ca 2؉ -saturated) states in reconstituted binary (cTnCcTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca 2؉ . Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca 2؉ -induced increases of >8 Å. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca 2؉ activation causes residues within cTnI-I to switch from a ␤-turn/ coil to an extended quasi-␣-helical conformation as the actin-contacts are broken, whereas cTnI-R remains ␣-helical in both Mg 2؉ -and Ca 2؉ -saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg 2؉ -and Ca 2؉ -saturated states.The contractile state of cardiac muscle is regulated by a series of Ca 2ϩ and cross-bridge-dependent interactions among the thin filament proteins, including the subunits of troponin (Tn), 1 tropomyosin, and actin. Troponin is composed of the three subunits: a Ca 2ϩ -binding subunit (TnC), an inhibitory subunit (TnI), which when bound to actin, inhibits myosin ATPase and force generation in relaxed muscle, and a tropomyosin-binding subunit (TnT). Cardiac muscle activation is initiated by the binding of Ca 2ϩ to the single N-domain regulatory site of cTnC. This binding causes structural changes between cTnC and two critical regions of cTnI: the regulatory region (cTnI-R, residues 150 -165) and the inhibitory region (cTnI-I, residues 130 -149). During activation, cTnI-I dissociates from actin and associates with cTnC (1), and cTnI-R associates with an activation-exposed hydrophobic patch in the N-domain of cTnC (2). These changes, together with movement of tropomyosin on the thin filament (3) and changes in actin structure and dynamics (4), switch on the thin filament, permitting actin-myosin cross-bridge cycling and force development.Numerous studies have elucidated Ca 2ϩ -induced structural changes between TnI and the other thin filament proteins (5, 6). Less is known about Ca 2ϩ -induced structural changes within TnI itself. We recently reported that in fully reconstituted cTn, Ca 2ϩ binding to cTnC causes a 9-Å increase in the length of cTnI-I (7). This large extension of the inhibitory region apparently pulls this region away from actin and facilitates movement of the adjacent regula...

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Section: Fig 5 Confidence Estimates In the Recovered Mean Distancesmentioning

confidence: 99%

Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

Dong¹,

Robinson²,

Stagg

et al. 2003

Journal of Biological Chemistry

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“…TnI is capable of inhibiting actomyosin ATPase in the absence of other subunits, but Ca 2ϩ -dependent regulation requires TnC, TnT, and tropomyosin. The inhibitory region (skTnI 96-117) alone can fully inhibit actomyosin ATPase activity (7) possibly by binding either to actin or to TnC in the ''on'' or ''off'' states (8)(9)(10). The corresponding residues for the cardiac inhibitory region are 129-150 because of a unique Ϸ32 residue N-terminal extension of cTnI.…”

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confidence: 99%

Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance

Brown

Sale

Hills

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Site-directed spin labeling EPR (SDSL-EPRtroponin I ͉ spin labels ͉ Fourier transform electron paramagnetic resonance ͉ DEER ͉ dipolar R egulation of striated muscle contraction is associated with Ca 2ϩ -dependent structural transitions in the muscle thin filament, which is composed of the troponin complex, tropomyosin, and actin. Troponin is composed of three components: TnC, which binds Ca 2ϩ ; TnI, which inhibits actomyosin activity; and TnT, which anchors TnC and TnI to tropomyosin. Muscle contraction is initiated by the binding of Ca 2ϩ to the N-lobe regulatory sites of TnC. The N lobe then undergoes a structural transition from a closed to an open form, which then facilitates the release of the inhibitory region of TnI from actin, binding to TnC and stimulation of the actomyosin ATPase. The mechanism of this signaling pathway is still tentative (for review, see ref. 1).Crystal and NMR structures are available for TnC and several complexes of TnC with TnI fragments. The crystal structure of TnC reveals a dumbbell-shaped protein consisting of two globular domains, joined by a 22-residue central ␣-helix (2-4). Each domain contains a hydrophobic cleft and two helix-loop-helix EF-hand metal binding motifs; two high-affinity Ca 2ϩ ͞Mg 2ϩ sites in the C-terminal domain (sites III and IV) and two low-affinity Ca 2ϩ specific sites in the N-terminal domain (sites I and II). In skeletal TnC, sites III and IV are permanently occupied by Mg 2ϩ and facilitate the structural binding of TnC to the contractile apparatus (5). Binding of Ca 2ϩ to sites I and II is the physiological trigger for muscle contraction. In cardiac TnC, binding site I is inactive and Ca 2ϩ binding to site II does not induce as large a structural change as observed in skeletal TnC (6). TnI is capable of inhibiting actomyosin ATPase in the absence of other subunits, but Ca 2ϩ -dependent regulation requires TnC, TnT, and tropomyosin. The inhibitory region (skTnI 96-117) alone can fully inhibit actomyosin ATPase activity (7) possibly by binding either to actin or to TnC in the ''on'' or ''off'' states (8-10). The corresponding residues for the cardiac inhibitory region are 129-150 because of a unique Ϸ32 residue N-terminal extension of cTnI.Structural information for the intact troponin complex is limited to low-resolution neutron diffraction and electron microscopy studies (11-13), though several high-resolution structures of TnC with bound TnI peptides are available. Two computational models have been recently proposed for the binary complex of TnC and TnI (14,15). Both models have TnI and TnC in an antiparallel arrangement with multiple interaction sites between the two subunits. NMR, crystallography, and neutron scattering was used by Tung et al. (15) to develop a computational model of the binary complex in which TnI winds around TnC in either a left-handed manner (model ''L'') or a right-handed manner (model ''R''). In both structures, the inhibitory region of TnI is modeled as a flexible ␤-hairpin in close proximity to the central helix of TnC. ...

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“…The equilibrium conformation of the regulatory region from cardiac muscle (3) and those of the skeletal regulatory region (4) and inhibitory region (5-7) have been reported. The interactions of these regions with skeletal TnC and actin have also been studied by FRET (8,9) and cross-linking (10,11). These studies have provided structural information on the thin filament proteins in the deactivated and activated states of the thin filament, in both skeletal and cardiac isoforms of the proteins, and insights on Ca 2ϩ -induced structural alterations that occur in the troponin complex upon activation.…”

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confidence: 99%

Kinetics of Conformational Transitions in Cardiac Troponin Induced by Ca2+ Dissociation Determined by Förster Resonance Energy Transfer

Dong

Robinson

Xing

et al. 2003

Journal of Biological Chemistry

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Upon Ca2؉ activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used Fö rster resonance energy transfer in Ca 2؉ titration and stoppedflow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca 2؉ dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca 2؉ from cTnC occurs in one step (t1 ⁄2 ϳ 5 ms), and this transition is not affected by Ca 2؉ release from the C-domain. The other two transitions triggered by Ca 2؉ dissociation are biphasic with the fast phase (t1 ⁄2 ϳ 5 ms) correlated with Ca 2؉ release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca 2؉ (t1 ⁄2 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca 2؉ switching between activation and deactivation of cardiac muscle contraction. Muscle activation involves Ca2ϩ -dependent changes in the structures of several proteins and protein-protein interactions within the thin filament. These proteins include the subunits of the heterotrimeric troponin complex (Tn), 1 tropomyosin, and actin. TnC is the Ca 2ϩ -binding subunit, TnI is the inhibitory subunit that binds to actin and inhibits actomyosin ATPase and force generation in relaxed muscle, and TnT anchors the three-subunit complex to tropomyosin on the actin filament. The initial activation step is the binding of activator Ca The Ca 2ϩ -linked molecular switch between relaxed and activated muscle is believed to be the inhibitory and regulatory regions of TnI. The switching mechanism is based on Ca 2ϩ -induced tertiary structure changes in the TnC N-domain coupled to secondary structure changes in the two TnI regions. In cardiac TnI (cTnI), the inhibitory region (cTnI-I) and the regulatory region (cTnI-R) span residues 130 -149 and 150 -165, respectively. The equilibrium conformation of the regulatory region from cardiac muscle (3) and those of the skeletal regulatory region (4) and inhibitory region (5-7) have been reported. The interactions of these regions with skeletal TnC and actin have also been studied by FRET (8, 9) and cross-linking (10, 11). These studies have provided structural information on the thin filament proteins in the deactivated and activated states of the thin filament, in both skeletal and cardiac isoforms of the proteins, and insights on Ca 2ϩ -induced structural alterations that occur in the troponin complex upon activation. These insights are central to our understanding of the activation mechanism. Because activation and deactivation...

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Calcium-Induced Movement of Troponin-I Relative to Actin in Skeletal Muscle Thin Filaments

Cited by 133 publications

References 20 publications

Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I

Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance

Kinetics of Conformational Transitions in Cardiac Troponin Induced by Ca2+ Dissociation Determined by Förster Resonance Energy Transfer

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