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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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Data from MD simulations.(a) The opening and closing of the cTnC N-domain was monitored by measuring the distance between the Cα of cTnC residues 13 and 51. The residues 13 and 51 are located on helix A of cTnC and on the linker region of helices B and C, respectively. In the Ca2+-free state the distance between the two residues decreased because the cTnC N-domain hydrophobic pocket closed (due to the loss of Ca2+ from the cTnC site 2). In the Ca2+-saturated state, the distance between these two residues increased as they moved away from each other because the hydrophobic pocket opened (due to Ca2+ in cTnC site 2). The Y-axis represents the distance (in nanometers) between the cTnC residues 13 and 51. (b) Depicts that RMSD of the protein in the Ca2+-saturated and Ca2+-free states. (c) The root mean square fluctuations of the cardiac troponin complex was calculated after allowing the initial 2 ns for equilibration. In the graph the C-alphas from 1–161 pertain to cTnC, 162–249 pertain to cTnT, 250–442 pertain to cTnI. Fluctuations of more than 3 Å are observed between C-alphas 162–177 in both the Mg2+ (Ca2+ free) and Ca2+ saturated states. This pertains to cTnT N-terminal helix H1 (C-alpha 162–177 in the graph pertain to residues 202–217 in the crystal structure). Fluctuations are also observed towards the C-terminal end of cTnT helix H2 in the Mg2+ state (C-alpha 234–249 in the graph pertain to cTnT residues 273–288 in the crystal structure). Towards the end of the X-axis we can see that the C-terminal end of cTnI experiences fluctuations in both the biochemical states. This pertains to the cTnI-Md.
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pone-0087135-g003: Data from MD simulations.(a) The opening and closing of the cTnC N-domain was monitored by measuring the distance between the Cα of cTnC residues 13 and 51. The residues 13 and 51 are located on helix A of cTnC and on the linker region of helices B and C, respectively. In the Ca2+-free state the distance between the two residues decreased because the cTnC N-domain hydrophobic pocket closed (due to the loss of Ca2+ from the cTnC site 2). In the Ca2+-saturated state, the distance between these two residues increased as they moved away from each other because the hydrophobic pocket opened (due to Ca2+ in cTnC site 2). The Y-axis represents the distance (in nanometers) between the cTnC residues 13 and 51. (b) Depicts that RMSD of the protein in the Ca2+-saturated and Ca2+-free states. (c) The root mean square fluctuations of the cardiac troponin complex was calculated after allowing the initial 2 ns for equilibration. In the graph the C-alphas from 1–161 pertain to cTnC, 162–249 pertain to cTnT, 250–442 pertain to cTnI. Fluctuations of more than 3 Å are observed between C-alphas 162–177 in both the Mg2+ (Ca2+ free) and Ca2+ saturated states. This pertains to cTnT N-terminal helix H1 (C-alpha 162–177 in the graph pertain to residues 202–217 in the crystal structure). Fluctuations are also observed towards the C-terminal end of cTnT helix H2 in the Mg2+ state (C-alpha 234–249 in the graph pertain to cTnT residues 273–288 in the crystal structure). Towards the end of the X-axis we can see that the C-terminal end of cTnI experiences fluctuations in both the biochemical states. This pertains to the cTnI-Md.

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)

Data from MD simulations.(a) The opening and closing of the cTnC N-domain was monitored by measuring the distance between the Cα of cTnC residues 13 and 51. The residues 13 and 51 are located on helix A of cTnC and on the linker region of helices B and C, respectively. In the Ca2+-free state the distance between the two residues decreased because the cTnC N-domain hydrophobic pocket closed (due to the loss of Ca2+ from the cTnC site 2). In the Ca2+-saturated state, the distance between these two residues increased as they moved away from each other because the hydrophobic pocket opened (due to Ca2+ in cTnC site 2). The Y-axis represents the distance (in nanometers) between the cTnC residues 13 and 51. (b) Depicts that RMSD of the protein in the Ca2+-saturated and Ca2+-free states. (c) The root mean square fluctuations of the cardiac troponin complex was calculated after allowing the initial 2 ns for equilibration. In the graph the C-alphas from 1–161 pertain to cTnC, 162–249 pertain to cTnT, 250–442 pertain to cTnI. Fluctuations of more than 3 Å are observed between C-alphas 162–177 in both the Mg2+ (Ca2+ free) and Ca2+ saturated states. This pertains to cTnT N-terminal helix H1 (C-alpha 162–177 in the graph pertain to residues 202–217 in the crystal structure). Fluctuations are also observed towards the C-terminal end of cTnT helix H2 in the Mg2+ state (C-alpha 234–249 in the graph pertain to cTnT residues 273–288 in the crystal structure). Towards the end of the X-axis we can see that the C-terminal end of cTnI experiences fluctuations in both the biochemical states. This pertains to the cTnI-Md.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087135-g003: Data from MD simulations.(a) The opening and closing of the cTnC N-domain was monitored by measuring the distance between the Cα of cTnC residues 13 and 51. The residues 13 and 51 are located on helix A of cTnC and on the linker region of helices B and C, respectively. In the Ca2+-free state the distance between the two residues decreased because the cTnC N-domain hydrophobic pocket closed (due to the loss of Ca2+ from the cTnC site 2). In the Ca2+-saturated state, the distance between these two residues increased as they moved away from each other because the hydrophobic pocket opened (due to Ca2+ in cTnC site 2). The Y-axis represents the distance (in nanometers) between the cTnC residues 13 and 51. (b) Depicts that RMSD of the protein in the Ca2+-saturated and Ca2+-free states. (c) The root mean square fluctuations of the cardiac troponin complex was calculated after allowing the initial 2 ns for equilibration. In the graph the C-alphas from 1–161 pertain to cTnC, 162–249 pertain to cTnT, 250–442 pertain to cTnI. Fluctuations of more than 3 Å are observed between C-alphas 162–177 in both the Mg2+ (Ca2+ free) and Ca2+ saturated states. This pertains to cTnT N-terminal helix H1 (C-alpha 162–177 in the graph pertain to residues 202–217 in the crystal structure). Fluctuations are also observed towards the C-terminal end of cTnT helix H2 in the Mg2+ state (C-alpha 234–249 in the graph pertain to cTnT residues 273–288 in the crystal structure). Towards the end of the X-axis we can see that the C-terminal end of cTnI experiences fluctuations in both the biochemical states. This pertains to the cTnI-Md.
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