Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction

Tripet, Brian; Eyk, Jennifer E. Van; Hodges, Robert S.

doi:10.1006/jmbi.1997.1200

Cited by 197 publications

(250 citation statements)

References 72 publications

Supporting

Mentioning

231

Contrasting

Order By: Relevance

“…A synthetic peptide consisting of residues 128 -148 was able to bind specifically to the actin-Tm filament and could induce a weak inhibitory activity on its own. Truncation of residues 140 -148 completely abolished the inhibitory effect of this region when compared with TnI-(96 -115), suggesting that region 140 -148 of TnI presumably contains the second actin-Tm-binding site (17). This is in agreement with Farah et al (18), who demonstrated that residues 116 -156 are important for the Ca 2ϩ regulation of actomyosin ATPase activity.…”

supporting

confidence: 87%

“…Sequence 104 -115 of TnI has been shown to share both the actin-Tm-and TnC-binding sites. Studies of Tripet et al (17) suggest that the region of TnI that follows the inhibitory sequence (residues 96 -116) contains additional actin-Tm-and TnC-binding sites. A synthetic peptide consisting of residues 128 -148 was able to bind specifically to the actin-Tm filament and could induce a weak inhibitory activity on its own.…”

mentioning

confidence: 99%

“…The recombinant fragment TnI-(1-116) failed to inhibit ATPase activity in the absence of Ca 2ϩ compared with other fragments (TnI-(1-156) and TnI-(103-182)) that were able to regulate actomyosin ATPase activity in a Ca 2ϩ -dependent manner (18). Furthermore, Tripet et al (17) demonstrated that residues 116 -131 are not important for inhibition, but are critical for the interaction with TnC and designated this region to be the second TnC-binding site (17). Several studies have shown that residues 96 -116 of TnI are primarily responsible for the binding to the COOH-terminal domain of TnC and residues 117-148 for the binding of TnI to the NH 2 -terminal domain of TnC (19,20).…”

mentioning

confidence: 99%

See 2 more Smart Citations

The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

Szczêsna

Zhang

Zhao

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.Contraction of skeletal muscle is initiated by the binding of Ca 2ϩ to the regulatory sites of troponin (Tn) 1 C, the Ca 2ϩ -binding subunit of Tn, which causes an interaction with TnI, the inhibitory subunit of Tn, releasing its inhibition of actomyosin ATPase activity. For full biological function, Tn requires a third subunit, TnT, which anchors the TnI⅐TnC complex to the actin-tropomyosin (Tm) filaments and also plays a role in the Ca 2ϩ -mediated activation of actomyosin ATPase activity and/or contraction (1-5).The inhibitory function of Tn has been studied in many different ways, yet the structure-function of the inhibitory region of TnI, responsible for this, is still under investigation. The role of the putative inhibitory region of TnI, which consists of 21 amino acids (residues 96 -116), has been studied previously utilizing synthetic peptides as well as proteolytic and recombinant fragments of TnI. The cyanogen bromide fragment (CN4) of TnI (residues 96 -116) was originally found to possess all of the inhibitory properties of intact TnI (6). Studies with synthetic peptides have demonstrated that residues 105-114 represent the minimal sequence necessary to produce inhibition of actomyosin ATPase activity and to retain TnC binding (7-10); however, the CN4 fragment (TnI-(96 -116)) has been shown to have ...

show abstract

supporting

confidence: 87%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

Szczêsna

Zhang

Zhao

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…TnC would lead to a more elongated configuration of the troponin complex [36][37][38]. There are multiple interactions between TnI and TnC, involving regions located in the N-terminal hydrophobic cleft (residues 51-62), central helix (residues 89-100), and the C-terminal hydrophobic cleft (residues 127-138) of TnC and regions located in the N-terminal part (residues 1-47) and the C-terminal part (residues 96-117 and 118-129) of TnI [3,13,17,39,40].…”

Section: Discussionmentioning

confidence: 99%

“…Based on several experimental approaches, such as crystallography [1][2][3], NMR [4][5][6], neutron scattering [7,8], chemical crosslinking [9-11] and fluorescence resonance energy transfer [12,13], a structural model of the TnC-TnI complex was proposed in which TnI winds around TnC in either a lefthanded manner (Model ÔLÕ) or a right-handed manner (Model ÔRÕ) [14]. According to these models, five adjoining segments of TnI, segment I-V, correspond to residues 3-33, [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114] respectively. Segment…”

mentioning

confidence: 99%

Calcium‐dependent protein–protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I

Liou¹,

Chen²

2003

European Journal of Biochemistry

View full text Add to dashboard Cite

The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca 2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)-iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca 2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca 2+ -dependent myofibrillar ATPase. The binding of Ca 2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca 2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction.Keywords: proximity relationships; cysteine residues; pyrene-containing fluorophores; monomer fluorescence; excimer fluorescence. Ca 2+ activation of striated muscle is mediated by the troponin complex. Troponin is composed of three subunits: troponin C (TnC), a Ca 2+ binding subunit; troponin I (TnI), an inhibitory subunit and troponin T (TnT), a subunit which binds the complex to tropomyosin. Based on several experimental approaches, such as crystallography [1][2][3], NMR [4][5][6], neutron scattering [7,8], chemical crosslinking [9-11] and fluorescence resonance energy transfer [12,13], a structural model of the TnC-TnI complex was proposed in which TnI winds around TnC in either a lefthanded manner (Model ÔLÕ) or a right-handed manner (Model ÔRÕ) [14]. According to these models, five adjoining segments of TnI, segment I-V, correspond to residues 3-33, [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][95][96][97][98][99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114] respectively. Segment

show abstract

Structural mapping of single cysteine mutants of cardiac troponin I

et al. 2000

View full text Add to dashboard Cite

The global conformation of cardiac muscle troponin I (cTnI) was investigated with single‐cysteine mutants by using a combination of sulfhydryl reactivity and fluorescence resonance energy transfer (FRET) to determine cysteine accessibility and intersite distances. The reactivity was determined with a fluorescent reagent for its reaction with cysteine residues singly located at positions 5, 40, 81, 98, 115, 133, 150, 167, and 192. FRET measurements were made by using the endogenous single Trp‐192 as the energy donor and an acceptor probe covalently attached to the cysteines as energy acceptor. The results suggest an open and extended conformation of cTnI with a large curvature in which the cysteines are highly exposed to the solvent. These conformational features are largely retained in the segment between residues 40 and 192 upon phosphorylation at Ser‐23 and Ser‐24. The sulfhydryl groups of the Cys‐133 and Cys‐150 of the cTnI incorporated into the binary cTnC‐cTnI and fully reconstituted troponin complexes experience large reduced exposure resulting from the binding of Ca2+ to the regulatory site of cTnC, suggesting that key regions of cTnI involved in activation become highly shielded upon activation. In the cTnC‐cTnI complex, every intramolecular distance in the cTnI is lengthened and the overall conformation of the bound cTnI remains elongated with reduced exposure for the cysteines. The global conformation of the troponin C‐troponin I complex from cardiac muscle has an elongated shape with constrained flexibility. The highly flexible nature of the N‐terminal extension of cTnI is preserved in the complex, suggesting that this segment of cTnI is either not bound or only loosely bound to the C‐domain of cTnC. Proteins 2000;41:438–447. © 2000 Wiley‐Liss, Inc.

show abstract

Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction

Cited by 197 publications

References 72 publications

The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

Calcium‐dependent protein–protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I

Structural mapping of single cysteine mutants of cardiac troponin I

Contact Info

Product

Resources

About