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

Abstract

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. ...

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

“…The conformation and orientation of cTnI [128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147] in this structure are similar to those reported from an electron spin labeling work which shows that a section of the inhibitory region (cTnI 129-137 ) displays a helical profile, with the C-terminal residues 139-145 displaying no discernible secondary structure characteristics [37]. While most of the inhibitory region of cTnI is not visualized in the cTnC·3Ca 2+ ·cTnI ·cTnT 182-288 structure, the conformation and orientation of the N-terminal and switch regions of cTnI are in good agreement with those observed from the binary complexes.…”

The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner

“…Alternatively, unbinding of the m-domain from actin could free the m-domain for interactions with other ligands, such as myosin S1 or S2, myosin light chains (32,42), or thin filament regulatory proteins. Such interactions could affect cross-bridge cycling kinetics by influencing the position or docking of myosin S1 heads onto the thin filament or by activating the thin filament in a manner analogous to the way in which TnI unbinding actin promotes thin filament activation (43,44). and C2.…”

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

“…In the intact thin filament, sTnI133 (cTnI167) is observed to move by 23.6 Å relative to actin filament, 2 and this movement is predominantly in the plane of the actin surface. N-cTnI-I is believed to remain fixed to cTnT/C-cTnC (19,20), and the proximity of the C-and N-cTnC domains is essentially unaltered by Ca 2ϩ addition (33). Assuming a relatively fixed position of N-TnC relative to the actin filament (34), the 19.7 Å 2 J. M. Robinson, W. J. Dong, and H. C. Cheung, unpublished result.…”

“…The Mg 2ϩ -saturated state was represented as a ␣-helix (residues 1-9)/␤-turn (residues 10 -21)/␣-helix (residues 22-39). Model residues 1-9 correspond to the N terminus of the inhibitory region (TnI-I), believed to form a stable ␣-helical coiled-coil with TnT (19,20). Residues 10 -21, corresponding to the C terminus of cTnI-I, associate with the actin filament in the Mg 2ϩ -saturated state.…”

“…Meanwhile, the N-terminal portion of cTnI-I associates with the C-domain of cTnC. This arrangement requires that the C-terminal portion of cTnI-I, which is accessible to solvent (19), bridges the N-and C-terminal cTnC domains, running roughly anti-parallel to the cTnC linker region. In the Mg 2ϩ -saturated state, cTnI-R remains ␣-helical.…”