Conformational Changes of Troponin C Within the Thin Filaments Detected by Neutron Scattering

Matsumoto, Fumiko; Makino, Kouji; Maéda, Kayo; Patzelt, Heiko; Maéda, Yuichiro; Fujiwara, Satoru

doi:10.1016/j.jmb.2004.07.086

Cited by 26 publications

(21 citation statements)

References 55 publications

(53 reference statements)

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“…For the inhibitory region, on the other hand, the crystal structure of the core domain of fsTn complex in the presence of Ca 2ϩ showed that it interacts with the central helical region (D/E helix) of TnC (9). For cardiac Tn, the D/E helix of TnC was disordered, and the inhibitory region was not visible in the crystal structure of the core domain of cTn complex (51), whereas small angle x-ray diffractions of the cardiac thin filaments indicate an extended structure of the D/E helix region of TnC (52). These observations indicate flexibility in the linker region of the Tn complex and suggest a relatively weak interaction between the inhibitory region of TnI and the central helix of TnC.…”

Section: Discussionmentioning

confidence: 99%

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

Kobayashi

Patrick

Kobayashi

2009

Journal of Biological Chemistry

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In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca 2؉ concentration. Tn is composed of TnC, TnI, and TnT. Ca 2؉ binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca 2؉ activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca 2؉ , which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca 2؉ NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca 2؉ , there was little difference in the activation of ATPase activity between these preparations.Striated muscle thin filaments exist in equilibrium among multiple states. Ca 2ϩ binding to the regulatory domain of troponin C (TnC) 2 along the thin filaments and strong crossbridge interactions with thick filaments are thought to shift the equilibrium. Ca 2ϩ binds to the regulatory domain of TnC, which regulates the interaction of troponin I (TnI) with actintropomyosin (Tm) and TnC (1-3). In the thin filaments, the inhibitory region of TnI (residues 104 -115 of rabbit fast skeletal TnI (fsTnI) or 138 -149 of mouse cardiac TnI (cTnI)) undergoes a structural transition depending on the Ca 2ϩ state of TnC (4, 5). In the absence of Ca 2ϩ at the regulatory site(s) of TnC, the inhibitory region interacts with actin to prevent activation of myosin ATPase activity. When Ca 2ϩ binds to the regulatory site(s) of TnC, the switch region of TnI, which is located at the C terminus of the inhibitory region, interacts with the newly exposed hydrophobic patch of the N-terminal regulatory domain of . This interaction causes the removal of the inhibitory region and the second actin-Tm binding region of TnI from the actin surface and allows actin to interact with myosin. In the presence of Ca 2ϩ at the regulatory sites of TnC, the inhibitory region and the central helical region of TnC are mutually stabilized, according to the recent x-ray crystal structure of the core domain of the fsTn complex (9). The sequence variations in the N-terminal and the C-terminal regions of TnT, another component of the Tn complex, are known to alter the Ca 2ϩ sensitivity of myofilament activity (10, 11). In addition, TnT is involved in the Ca 2ϩ -dependent interaction of the Tn complex with actin-...

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Section: Discussionmentioning

confidence: 99%

Ala Scanning of the Inhibitory Region of Cardiac Troponin I

Kobayashi

Patrick

Kobayashi

2009

Journal of Biological Chemistry

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“…F-actin was purified from chicken pectoralis muscle (54) and native thin filaments from porcine cardiac muscle (55,56). Bovine cardiac tropomyosin and troponin were produced as previously described (57).…”

Section: Methodsmentioning

confidence: 99%

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

Mun

Previs

et al. 2014

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

138

207

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Significance Myosin-binding protein C (MyBP-C) is a component of myosin filaments, one of the two sets of contractile elements whose relative sliding is the basis of muscle contraction. In the heart, MyBP-C modulates contractility in response to cardiac stimulation; mutations in MyBP-C lead to cardiac disease. The mechanism by which MyBP-C modulates cardiac contraction is not understood. Using electron microscopy and a light microscopic assay for filament sliding, we demonstrate that MyBP-C binds to the other set of filaments, containing actin and the regulatory component, tropomyosin. In so doing, it displaces tropomyosin from its inhibitory position to activate actin filament interaction with myosin, promoting filament sliding. These findings provide insights into the molecular basis of heart function.

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“…22 dTnC was prepared and reconstituted into NTF as described. 8 Sols of NTF and dTnC-RTF in 2 H 2 O containing 40 mM Tris-HCl (pD 7.5), 1 mM MgCl 2 , 1 mM 2-mercaptoethanol, and 1 mM EGTA (or 2 mM CaCl 2 added for the state in the presence of Ca 2+ ), were poured into quartz capillaries with a diameter of 3 mm, sealed, and oriented by placing them in a magnetic field of 18 T. 9 The samples were checked with a polarized microscopy and confirmed that an oriented region had a length of about 3 mm near the bottom of the capillary. Neutron fiber diffraction patterns of these oriented samples were measured with the small-angle scattering instrument, SANS-J, installed in the guide hall of the reactor JRR-3M in Japan Atomic Energy Agency, Ibaraki, Japan.…”

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confidence: 99%

“…6,7 We showed from the neutron scattering measurements of the thin filaments containing deuterated TnC (dTnC) under the condition where the protonated components were rendered "invisible" to neutrons by the contrast matching technique that TnC assumes extended dumbbell-like structures and moves toward the filament axis by binding of Ca 2+ . 8 More detailed in situ structural information such as orientation of TnC should contribute to elucidate the interactions between Tn-subunits. Such information was not available from the neutron scattering measurements on solution samples because of spatial averaging.…”

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confidence: 99%

“…(b) Construction of the projected density distribution of TnC. As TnC was found to assume an extended dumbbell-like structure within the thin filaments, 8 TnC was modeled as two three-dimensional isotropic Gaussian functions connected by a cylindrical linker. The N-terminal domain (residues 1-86) of TnC was estimated from the atomic coordinates to have a radius of gyration (R g ) of 12.1 Å (the average of the values calculated from 1J1D 4 and 1J1E 4 under the condition of the protonated protein in 100% 2 H 2 O with 80% exchange of H and 2 H 11 ) in the presence of Ca 2+ , and 11.7 Å (the value calculated from 1SPY 28 ) in the absence of Ca 2+ .…”

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confidence: 99%

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