ROLE OF THE C-TERMINUS MOBILE DOMAIN OF CARDIAC TROPONIN I IN THE REGULATION OF THIN FILAMENT ACTIVATION IN SKINNED PAPILLARY MUSCLE STRIPS

The C-terminus mobile domain of cTnI (cTnI-MD) is a highly conserved region which stabilizes the actin-cTnI interaction during the diastole. Upon Ca2+-binding to cTnC, cTnI-MD participates in a regulatory switching that involves cTnI to switch from interacting with actin toward interacting with the Ca-regulatory domain of cTnC. Despite many studies targeting the cTnI-MD, the role of this region in the length-dependent activation of cardiac contractility is yet to be determined. The present study investigated the functional consequences of losing the entire cTnI-MD in cTnI(1-167) truncation mutant, as it was exchanged for endogenous cTnI in skinned rat papillary muscle fibers. The influence of cTnI-MD truncation on the extent of the N-domain of cTnC hydrophobic cleft opening and the steady-state force as a function of sarcomere length (SL), cross-bridge state, and [Ca2+] was assessed using the simultaneous in-situ time-resolved FRET and force measurements at short (1.8 µm) and long (2.2µm) SLs. Our results suggest that the cTnI-MD governs the equilibrium position of tropomyosin on actin filament at both relaxed and activated states and mediates the level of thin filament activation. Our results also suggest that cTnI-MD transmits the effects of SL change to the core of troponin complex.

FRET distance measurements within the cardiac troponin complex.

<p>The FRET distances measured between donor and acceptor probes in the Ca²⁺-saturated and the Ca²⁺-free states are tabulated. The half-widths are parenthesized. The FRET distances and restraints were applied as NOE restraints <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone.0087135-Sheldahl1" target="_blank">[49]</a> between the Cα's of the participating amino acids. In columns 3,4 and 5,6 the experimentally measured FRET distance is tabulated along with the distance between the Cα's of the participating amino acids in the modeled structures, in the Ca²⁺-saturated states and Ca²⁺-free states respectively. In the FRET analysis, due to the ambiguity in the value of the dipole–dipole orientation factor between energy donor molecules and energy acceptor molecules and due to the dimensions of the probes attached by linkers to the side chains of the amino acid residues, the measured distance will have an uncertainty of ±10% <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone.0087135-dosRemedios1" target="_blank">[53]</a>. Although the length of probe linkers is ∼10 Å, the linker is not unidirectional but folds randomly (during folding and rotation the probe and can acquire length of ∼7 Å). Based on these above factors there is good correlation between the measured FRET distance and the model. The italicized numbers in the third column pertain to the distance between the C-alphas in the crystal structure (1J1E). Compared to X-ray crystallography technique, FRET is a low resolution structural tool, and it does not have the lattice constraints that would be present in X-ray determined structure. However, FRET can acquire structural information in a more physiological environment, particularly with time-resolved approach (as we used here) it can provide dynamic information (represented by HW of the distance distribution) associated with each measured distance. Broad distributions of our FRET distances listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone-0087135-t001" target="_blank">Table 1</a> suggest that troponin exhibits a much dynamic structure in solution than in crystal. Therefore some discrepancies in the mean FRET distances with respect to the distances measured in X-ray structure would be expected. If we consider the structural dynamics (large HWs) observed in solution samples, these differences are in reasonable range.</p

Electrostatic surface analysis of the N-domain of cTnC.

<p>Depicts the N-domain of cTnC in the Ca²⁺-free state (figures a through c) and Ca²⁺-saturated states (figures d through f). The cTnC N-domain helices A through D are colored as red, green, cyan and blue respectively. (a) Transparent rendering of the electrostatic surface is shown with the protein in cartoon. (b) In the Ca²⁺-free state the loss of regulatory Ca²⁺ caused the rearrangement of helices B and C. These helices are no longer orthogonal to each other but nearly parallel. This resulted in a breach in the hydrophobic pocket that surrounded the cTnI-Rr (pointed out by the arrow). (c) The cTnC N-domain hydrophobic pocket when viewed from below the cTnC N-domain. This view shows the hydrophobic environment in which the cTnI-Rr is located. The arrows points to the gap in the hydrophobic pocket. (d) Transparent rendering of the electrostatic surface with the protein rendered as cartoon. (e) In the Ca²⁺-saturated state there is no breach in the hydrophobic pocket within which the cTnI-Rr is held. (f) The cTnC N-domain hydrophobic pocket in the Ca²⁺-saturated state when view from below.</p

Ca²⁺-free state structure of the cardiac troponin complex.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone-0087135-g005" target="_blank">Fig. 5c</a>. (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.</p

Electrostatic surface analysis of the cTnC and cTnI.

<p>Depicts the electrostatics in the vicinity of cTnC helix E in the Ca²⁺-free state after 9.5 ns of simulation. The hydrophobic, negative and positive surfaces are colored white, red and blue respectively. (a) The thick arrow points to the unfolded segment in helix E. The positively charged blue residues of cTnI-Ir are seen arching (pointed to by the dotted line with arrow head) towards the unfolded cTnC helix E (pointed to by thick black arrow). The amino acids sequence of the cTnI-Ir residues is 138-KFKRLPT and the sequence of the opposing cTnC residues are 92-KSEEEL. The predominantly negative (red) cTnC helix E is attracted to the positive region of cTnI-Ir. The unfolded helix E has adopted a “U” shape (pointed to by the thick black arrow). (b) The unfolded helix E is seen in concert with cTnI and cTnT. The cTnC Glu94 is attracted to cTnI Lys141 (not seen in picture), cTnC Glu95 is attracted to the nitrogen on Leu129 of cTnI and Arg142 of cTnI, and cTnC Glu96 is attracted to Arg 267 of cTnT.</p

FRET distance distribution between cTnT and cTnI.

<p>Distribution of the distances P(r) between cTnT residue 276 and cTnI residues 151, 160, and 167, determined in the reconstituted cTn complex at low Ca²⁺ (broken curve) and saturating Ca²⁺ (solid curve).</p

Secondary structure timeline of the cTnI-Rr/switch.

<p>The cTnI-Rr is held within the cTnC N-domain hydrophobic pocket. In the presence of regulatory Ca²⁺ the cTnI-Rr maintains a helical conformation. In the absence of regulatory Ca²⁺ the secondary structure is perturbed. The absence of the helical conformation would release the cTnI-Md to interact with actin in the Ca²⁺-free state whereas, the presence of the regulatory Ca²⁺ would refold the cTnI-Rr into a helix effectively retracting the cTnI-Md from actin.</p

Hydrogen bonds formed in the Ca²⁺-saturated state structure after 11.1 ns of simulations.

<p>The intra- and inter-molecular hydrogen bonds formed within the cTn complex in the Ca²⁺-saturated state after 11.1 ns of simulations.</p

The starting structure for MD simulations.

<p>The crystal structure (1J1E.pdb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087135#pone.0087135-Takeda1" target="_blank">[10]</a>) of the cTn complex at saturating Ca²⁺ is depicted with the N-terminal extension of cTnI (cTnI-Nxt) docked over the cTnC N-domain. The N-terminal extension of cTnI was docked to the cTn complex using Hex, a protein-protein docking program. The cTnT and cTnI are colored cyan and blue respectively. The cTnC helices N, A, B, C, D in the N-domain are colored, red, mustard, orange, yellow and lime green. The bound Ca²⁺ ions at sites 2, 3, and 4 are rendered as spheres and colored cyan.</p

Data from MD simulations.

<p>(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.</p