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Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints.

Jayasundar JJ, Xing J, Robinson JM, Cheung HC, Dong WJ - PLoS ONE (2014)

Bottom Line: In the presence of saturating Ca(2+) the above said phenomenon were absent.We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin.Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

View Article: PubMed Central - PubMed

Affiliation: Voiland School of Chemical Engineering and Bioengineering and The Department of Integrated Physiology and Neuroscience, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Cardiac troponin (cTn) is the Ca(2+)-sensitive molecular switch that controls cardiac muscle activation and relaxation. However, the molecular detail of the switching mechanism and how the Ca(2+) signal received at cardiac troponin C (cTnC) is communicated to cardiac troponin I (cTnI) are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET) measurements and molecular dynamic (MD) simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca(2+)-free and saturating Ca(2+) conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca(2+)-saturated structure, the absence of regulatory Ca(2+) perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca(2+), induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca(2+) the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

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Related in: MedlinePlus

Ca2+-free state structure of the cardiac troponin complex.The cTn structure after 9.52+-free state is depicted using CCP4MG version 2.7.3. (a) The cTn complex is positioned such as to see the dynamics of the cTnI-Md. The cTnI-Md is seen to have positioned itself close to the cTnC helix A. The cTnI-Rr held within the cTnC N-domain hydrophobic pocket has its secondary structure perturbed due to the closing of the hydrophobic pocket. (b) Depicts the cTnC N-domain wherein the loss of regulatory Ca2+ led to structural rearrangement of the helices B, C and D. The helices B and C are almost parallel to each other. (c) View of the N-terminal extension of cTnI above the cTn core domain complex. In this view the collapsed conformation of the cTnC N-domain hydrophobic pocket is well seen. It may be compared against the conformation of the open cTnC N-domain hydrophobic pocket in Fig. 5c. (d) The averaged structure of the cardiac troponin complex in the Ca2+ free state from 2 ns to 9.5 ns. The first two nanoseconds were given for the system to equilibrate. (e) The secondary structure of the troponin C in the presence of FRET distance restraints are calculated from 2 ns till 11.1 ns. The cTnC N-domain helices N (residues 4–11), A (residues 14–28), B (residues 38–47), C (residues 54–61) and D (residues 74–85) experienced considerable secondary structure evolution, but in contrast, the cTnC C-domain helices E (residues 94–104), F (residues 117–123), G (residues 130–140), and H (residues 150–157) are comparatively stable. Perturbation in the structure of helix D pulls and releases the D/E linker which in turn unfolds and refolds helix E. This fluctuations cause the D/E linker to alternate between flexible and rigid conformations that effectively helps release and retract the cTnI-Ir towards and away from actin in the absence and presence of Ca2+. The residue numbers associated with the helices of cTnC which are given within brackets were derived from the crystal structure 1J1E.pdb.
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pone-0087135-g004: Ca2+-free state structure of the cardiac troponin complex.The cTn structure after 9.52+-free state is depicted using CCP4MG version 2.7.3. (a) The cTn complex is positioned such as to see the dynamics of the cTnI-Md. The cTnI-Md is seen to have positioned itself close to the cTnC helix A. The cTnI-Rr held within the cTnC N-domain hydrophobic pocket has its secondary structure perturbed due to the closing of the hydrophobic pocket. (b) Depicts the cTnC N-domain wherein the loss of regulatory Ca2+ led to structural rearrangement of the helices B, C and D. The helices B and C are almost parallel to each other. (c) View of the N-terminal extension of cTnI above the cTn core domain complex. In this view the collapsed conformation of the cTnC N-domain hydrophobic pocket is well seen. It may be compared against the conformation of the open cTnC N-domain hydrophobic pocket in Fig. 5c. (d) The averaged structure of the cardiac troponin complex in the Ca2+ free state from 2 ns to 9.5 ns. The first two nanoseconds were given for the system to equilibrate. (e) The secondary structure of the troponin C in the presence of FRET distance restraints are calculated from 2 ns till 11.1 ns. The cTnC N-domain helices N (residues 4–11), A (residues 14–28), B (residues 38–47), C (residues 54–61) and D (residues 74–85) experienced considerable secondary structure evolution, but in contrast, the cTnC C-domain helices E (residues 94–104), F (residues 117–123), G (residues 130–140), and H (residues 150–157) are comparatively stable. Perturbation in the structure of helix D pulls and releases the D/E linker which in turn unfolds and refolds helix E. This fluctuations cause the D/E linker to alternate between flexible and rigid conformations that effectively helps release and retract the cTnI-Ir towards and away from actin in the absence and presence of Ca2+. The residue numbers associated with the helices of cTnC which are given within brackets were derived from the crystal structure 1J1E.pdb.

Mentions: The experimentally measured FRET distance distributions in the Ca2+-free state (Table 1), were applied as distance restraints and bond energies while performing distance restrained EM, distance restrained SA and distance restrained MD simulations. In addition to the FRET distances listed in Table 1, FRET distances and half-widths from previous studies were also used as distance restraints and bond-energies in these distance-restrained simulations. They were the distances between cTnC residues 13 and 51 [6], [18] and the distance between cTnI residues 5 and 192 determined in the presence and absence of Ca2+[22]. The Ca2+-free cardiac troponin complex was simulated for 9.5 ns under FRET distance restrains. The structure at the end of 9.5 ns is presented in Fig. 4. The simulated structure faithfully reproduced the closed cTnC N-domain hydrophobic pocket. The distance between residues 13 and 51 of cTnC decreased (Figure 3a) and this was consistent with previous experimental results [6], [18]. The root mean square deviation (RMSD) and root mean square fluctuations (RMSF) of the protein are presented in Figures 3b and 3c. Inspection of the simulated (Fig. 4a–c) and averaged structures (Fig. 4d) provided us detailed structural information on each of troponin subunit in the complex.


Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints.

Jayasundar JJ, Xing J, Robinson JM, Cheung HC, Dong WJ - PLoS ONE (2014)

Ca2+-free state structure of the cardiac troponin complex.The cTn structure after 9.52+-free state is depicted using CCP4MG version 2.7.3. (a) The cTn complex is positioned such as to see the dynamics of the cTnI-Md. The cTnI-Md is seen to have positioned itself close to the cTnC helix A. The cTnI-Rr held within the cTnC N-domain hydrophobic pocket has its secondary structure perturbed due to the closing of the hydrophobic pocket. (b) Depicts the cTnC N-domain wherein the loss of regulatory Ca2+ led to structural rearrangement of the helices B, C and D. The helices B and C are almost parallel to each other. (c) View of the N-terminal extension of cTnI above the cTn core domain complex. In this view the collapsed conformation of the cTnC N-domain hydrophobic pocket is well seen. It may be compared against the conformation of the open cTnC N-domain hydrophobic pocket in Fig. 5c. (d) The averaged structure of the cardiac troponin complex in the Ca2+ free state from 2 ns to 9.5 ns. The first two nanoseconds were given for the system to equilibrate. (e) The secondary structure of the troponin C in the presence of FRET distance restraints are calculated from 2 ns till 11.1 ns. The cTnC N-domain helices N (residues 4–11), A (residues 14–28), B (residues 38–47), C (residues 54–61) and D (residues 74–85) experienced considerable secondary structure evolution, but in contrast, the cTnC C-domain helices E (residues 94–104), F (residues 117–123), G (residues 130–140), and H (residues 150–157) are comparatively stable. Perturbation in the structure of helix D pulls and releases the D/E linker which in turn unfolds and refolds helix E. This fluctuations cause the D/E linker to alternate between flexible and rigid conformations that effectively helps release and retract the cTnI-Ir towards and away from actin in the absence and presence of Ca2+. The residue numbers associated with the helices of cTnC which are given within brackets were derived from the crystal structure 1J1E.pdb.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928104&req=5

pone-0087135-g004: Ca2+-free state structure of the cardiac troponin complex.The cTn structure after 9.52+-free state is depicted using CCP4MG version 2.7.3. (a) The cTn complex is positioned such as to see the dynamics of the cTnI-Md. The cTnI-Md is seen to have positioned itself close to the cTnC helix A. The cTnI-Rr held within the cTnC N-domain hydrophobic pocket has its secondary structure perturbed due to the closing of the hydrophobic pocket. (b) Depicts the cTnC N-domain wherein the loss of regulatory Ca2+ led to structural rearrangement of the helices B, C and D. The helices B and C are almost parallel to each other. (c) View of the N-terminal extension of cTnI above the cTn core domain complex. In this view the collapsed conformation of the cTnC N-domain hydrophobic pocket is well seen. It may be compared against the conformation of the open cTnC N-domain hydrophobic pocket in Fig. 5c. (d) The averaged structure of the cardiac troponin complex in the Ca2+ free state from 2 ns to 9.5 ns. The first two nanoseconds were given for the system to equilibrate. (e) The secondary structure of the troponin C in the presence of FRET distance restraints are calculated from 2 ns till 11.1 ns. The cTnC N-domain helices N (residues 4–11), A (residues 14–28), B (residues 38–47), C (residues 54–61) and D (residues 74–85) experienced considerable secondary structure evolution, but in contrast, the cTnC C-domain helices E (residues 94–104), F (residues 117–123), G (residues 130–140), and H (residues 150–157) are comparatively stable. Perturbation in the structure of helix D pulls and releases the D/E linker which in turn unfolds and refolds helix E. This fluctuations cause the D/E linker to alternate between flexible and rigid conformations that effectively helps release and retract the cTnI-Ir towards and away from actin in the absence and presence of Ca2+. The residue numbers associated with the helices of cTnC which are given within brackets were derived from the crystal structure 1J1E.pdb.
Mentions: The experimentally measured FRET distance distributions in the Ca2+-free state (Table 1), were applied as distance restraints and bond energies while performing distance restrained EM, distance restrained SA and distance restrained MD simulations. In addition to the FRET distances listed in Table 1, FRET distances and half-widths from previous studies were also used as distance restraints and bond-energies in these distance-restrained simulations. They were the distances between cTnC residues 13 and 51 [6], [18] and the distance between cTnI residues 5 and 192 determined in the presence and absence of Ca2+[22]. The Ca2+-free cardiac troponin complex was simulated for 9.5 ns under FRET distance restrains. The structure at the end of 9.5 ns is presented in Fig. 4. The simulated structure faithfully reproduced the closed cTnC N-domain hydrophobic pocket. The distance between residues 13 and 51 of cTnC decreased (Figure 3a) and this was consistent with previous experimental results [6], [18]. The root mean square deviation (RMSD) and root mean square fluctuations (RMSF) of the protein are presented in Figures 3b and 3c. Inspection of the simulated (Fig. 4a–c) and averaged structures (Fig. 4d) provided us detailed structural information on each of troponin subunit in the complex.

Bottom Line: In the presence of saturating Ca(2+) the above said phenomenon were absent.We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin.Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

View Article: PubMed Central - PubMed

Affiliation: Voiland School of Chemical Engineering and Bioengineering and The Department of Integrated Physiology and Neuroscience, Washington State University, Pullman, Washington, United States of America.

ABSTRACT
Cardiac troponin (cTn) is the Ca(2+)-sensitive molecular switch that controls cardiac muscle activation and relaxation. However, the molecular detail of the switching mechanism and how the Ca(2+) signal received at cardiac troponin C (cTnC) is communicated to cardiac troponin I (cTnI) are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET) measurements and molecular dynamic (MD) simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca(2+)-free and saturating Ca(2+) conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca(2+)-saturated structure, the absence of regulatory Ca(2+) perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca(2+), induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca(2+) the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

Show MeSH
Related in: MedlinePlus