Ca2+-induced conformational changes and protein-protein interactions within cardiac thin filament are the molecular basis of regulation of cardiac muscle function. An important mechanism for this regulation is that the transduction of the Ca2+ activation signal along the thin filament is modulated by strong crossbridge formation between myosin head and actin. This feature is clearly demonstrated in the present study of the kinetics of the opening of cTnC N-domain. This modulation is further fine tuned by phosphorylation of cTnI in response to pathological development or various demands of the heart. In the present work, we have shown that Ca2+ binding to the single regulatory site of cTnC is a major molecular interaction to convert its N-domain from a closed conformation to a relatively open conformation. In our previous studies, a given residue was replaced by tryptophan to serve as a donor of FRET to its acceptor attached to a specific cysteine. The two-cysteine marker used in the present study for the closed ↔ open conformational studies is a convenient FRET marker to monitor changes in intramolecular distances in cTnC that is reconstituted with other contractile proteins into physiologically relevant complexes without optical interference from endogenous tryptophans in these other proteins. Our results show that the two-cysteine marker is comparable to the previously used tryptophan-cysteine marker to report inter-site distances in cTnC reconstituted with tryptophanless proteins.
If the closed and open conformations of the cTnC N-domain are considered to be at equilibrium, Ca2+
binding to the domain shifts the equilibrium toward the open conformation. The ratio of the two conformations remains about the same in cTn (data not shown) and cTnTm. In regulated thin filaments, the FRET distance in Ca2+
-saturated N-domain (27.6 Å) is smaller than that in cTnTm (29.4 Å). This open N-domain of cTnC may accommodate the regulatory region of cTnI via a hydrophobic interaction between a hydrophobic patch in the N-domain and the cTnI regulatory region. Binding of strong S1 to the Ca2+
bound thin filament results in a longer distance (close to 30 Å, an increase of ca. 2.4 Å), shifting the closed ↔ open equilibrium and increasing the fraction of the open conformation (8
). The S1 binding disengages the inhibitory region of cTnI from actin, and the consequent change of the inhibitory region from a helix-coil-helix motif to an extended segment pushes the regulatory region toward the open cTnC N-domain. These events appear to be energetically favorable and thus promote stabilization of the open N-domain. Alternatively, the more open conformation may allow better contacts between the hydrophobic patch and the cTnI regulatory region, thus further stabilizing the open conformation. We do not have sufficient information to favor one or the other possibility. In the absence of bound S1 in the thin filament, binding of regulatory Ca2+
to cTnC is cooperative, likely due to the complex structural arrangement of the proteins in the filament and interactions among regulatory units (cTnI, cTnT, actin). Some of these interactions may be lost when the inhibitory region of cTnI is disengaged from actin. This disengagement could result in a loss of cooperativity in Ca2+
binding, but a gain in Ca2+
sensitivity (i.e., an increase of pCa50
The kinetics of FRET changes associated with Ca2+ binding differs from that associated with Ca2+ dissociation. The binding kinetics shows three phases, although the initial very fast phase with a considerable amplitude is not resolvable. The dissociation kinetics is biphasic with a very small loss in unresolvable FRET signal. Amplitudes in multi-phase kinetic transitions detected by the signal of a single probe are usually difficult to interpret, but in FRET-monitored kinetics each amplitude is quantitatively related to a change in energy transfer efficiency and, therefore, to a change in distance between two sites. The FRET kinetics associated with Ca2+ binding indicates that the Ca2+-induced opening of the cTnC N-domain occurs in three steps, and the kinetics associated with Ca2+ dissociation indicates that the closing of the N-domain is a two-step mechanism. The initial very fast step associated with domain opening is accompanied by the largest structural change in terms of the separation between two specific sites, followed by smaller structural changes in two slower steps. The rates of the two resolvable phases differ by a factor of about 10 in all three preparations. Phosphorylated cTnI has only a small effect on the difference between the two rates.
A previous kinetic study monitored by the fluorescence of a single environmentally sensitive probe attached to Cys35 of cTnC reported two transitions upon Ca2+
). These two transitions may correspond to the two resolved transitions in the present FRET-based kinetics for domain opening. The following summarizes the present kinetic results:
In this scheme, filled circle represents the closed conformation of the N-domain of cTnC in thin filaments, partially filled circle is partially open N-domain, and open circle is fully open N-domain. (Ca-TF)* represents an intermediate state of partially open conformation of the N-domain of cTnC. The initial binding of Ca2+
to cTnC is assumed to be a rapid equilibrium with equilibrium constant K0
, followed by multiple structural transitions. Ca2+
-induced opening involves movements of helices B and C away from the central helix resulting in a large change in the interhelical angle to accommodate the binding of the regulatory region of cTnI (5
). The observed multiple kinetic transitions may reflect these helical reorientations and movements to accommodate bound Ca2+
. Upon Ca2+
dissociation from the cTnC N-domain, domain closing occurs in two steps. These kinetic transitions may also be associated with changes of the reorientations of the helices B and C, but assignments of the kinetic rates to specific structural changes cannot be made in the present study.
The structural consequence of phosphorylation of cTnI on protein-protein interactions that occur within the cTn complex as well as conformational changes of cTnI have been extensively studied using NMR (22
) and FRET (21
). These studies suggest that the N-terminal extension of cTnI, most likely the residues immediately before the PKA phosphorylation sites, interact with the N-domain of cTnC. Phosphorylation of cTnI negatively affects the interactions. It has been proposed that shifting the open-closed equilibrium conformation in the N-domain toward the closed state by cTnI phosphorylation is likely the molecular basis for the decrease in Ca2+
sensitivity of thin filament activation. For example, a recent NMR study of the cTnC-cTnI binary complex has proposed a model for cTnI phosphorylation-induced conformational changes in cTnC (24
). This model suggests that the Ca2+
-induced open conformation of the N-domain adopts a partially closed structure in the complex when cTnI is phosphorylated by PKA and this partially closed conformation becomes more closed in the presence of both PKA and PKC phosphorylations.
The present steady-state and time-resolved FRET results show the distance between residues 13 and 51 of cTnC not to be affected by phosphorylation of cTnI regardless of whether actin or S1 is present. This finding appears not in accord with the proposed model (24
), suggesting that modulation of cTnI phosphorylation on thin filament activation is unlikely through a direct structural effect. FRET-based Ca2+
titration reveals a reduction in the Ca2+
sensitivity of the cTnC N-domain opening in the complexes. The observed cTnI phosphorylation-induced reduction of Ca2+
sensitivity is consistent with the notion that β-adrenergic stimulation reduces cardiac myofilament Ca2+
sensitivity. Kinetic results reveal that PKA phosphorylation of cTnI increase both the closing and open rate of conformational changes in the cTnC N-domain. The observed phosphorylation-induced kinetic effects on the cTnC N-domain structural transition are consistent with the role of β-adrenergic stimulation in the myocardium, i.e. increasing contractile force, enhancing heart rate and fastening relaxation of myocardial cells. Acceleration of the rate of relaxation is important for proper heart pumping function because it allows adequate time for diastolic filling of the ventricles against the raised heart rate during sympathetic stimulation. The present results would suggest that PKA phosphorylation of cTnI may exert its roles in cardiac systolic contraction and diastolic relaxation by modification of the kinetics of conformational transitions in cTnC.
In summary, we have used a two-cysteine FRET-based conformational marker to determine an inter-site distance in the regulatory N-domain of cTnC reconstituted into regulated thin filaments. Changes in this distance arising from binding and dissociation of regulatory Ca2+, strong binding of myosin S1, and phosphorylation of cTnI by PKA were determined from equilibrium and transient kinetic measurements. These changes demonstrate ligand-induced opening and closing of the cTnC N-domain. Phosphorylation of cTnI has negligible effects on the N-domain conformation regardless of the presence or absence of bound ligands, but it affects Ca2+ sensitivity and modifies the kinetics of opening and closing of the N-domain induced by binding of Ca2+ and S1. These results suggest that the mechanism of modulation of cardiac thin filament regulation by cTnI phosphorylation may be related to the altered kinetics of conformational transitions of the cTnC N-domain imposed by the phosphorylation.