Structural and Kinetic Effects of PAK3 Phosphorylation Mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca 2+, but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca 2+-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca 2+-induced changes in cTnI–cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC–cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca 2+ by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca 2+ dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI

Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments

Regulation of cardiac muscle function is initiated by binding of Ca 2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine tuned by phosphorylation of cTnI. The objective of the present study is to use a new Förster Resonance Energy Transfer-based structural marker to distinguish structural and kinetic effects of Ca 2+ binding, crossbridge interaction and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin and actin. Changes in the distance between Cys13 and Cys51 induced by Ca 2+ binding/dissociation were determined by FRET-sensed Ca 2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca 2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca 2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca 2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes

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

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

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

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

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