To understand the structural and functional significance of PAK3-mediated phosphorylation of cTnI, we used FRET approaches to investigate the effects of cTnI (S151E) on the Ca2+-induced structural transitions in cardiac thin filaments. Pseudo-phosphorylation of cTnI (S151E) had three major effects: 1) enhanced the interaction between the regulatory region of cTnI and cTnC, 2) increased the sensitivity of cardiac thin filaments to Ca2+, and 3) slowed kinetics of structural changes between cTnI-cTnC upon Ca2+ dissociation from troponin. Interestingly, these observed changes are similar to strong crossbridge mediated effects on thin filament.
The strengthened cTnI-cTnC interaction, caused by pseudo-phosphorylation of cTnI (S151E), was manifested by decreases in the distances between cTnI and cTnC. Results from the steady-state and time-resolved FRET measurements (, , and ) showed that the distances between the central helix of cTnC and the regulatory region of cTnI were significantly decreased in the presence of pseudo-phosphorylation of cTnI, regardless of the presence or absence of strongly-bound S1. Previous studies have shown that strong crossbridges formed between myosin heads and actin significantly decrease the distance between the inhibitory region of cTnI and the central helix of cTnC, leading to the enhancement cTnC-cTnI interaction1
. The enhanced cTnC-cTnI interaction is believed to be the result of feedback structural effect of strongly-bound S1 on the thin filament. The mechanism underlying the feedback effect is that the strongly-bound S1 effectively shifts Tm on the actin surface from the “closed” to the “open” position. The shift of Tm on actin surface weakens cTnI-actin interactions and favors Ca2+
-mediated interactions between cTnC and cTnI. This hypothesis is supported by the decrease in FRET distances between cTnC and cTnI when strongly-bound S1 is present ( and ). We believe that the structural effect induced by pseudo-phosphorylation of cTnI (S151E) is due to the phosphorylation-induced charge modification at the interface between cTnC and cTnI. It is known that the inhibitory region of cTnI is highly positively charged, especially at the junction between the inhibitory and the regulatory regions. In particular, there are three positively-charged arginine residues next to the phosphorylatable residue, Ser15110
. PAK3 phosphorylation-induced negative charges at Ser151 may potentially neutralize the overall positive surface charges at the junction between the regulatory and the inhibitory regions of cTnI. Such charge neutralization favors strong hydrophobic interaction between the regulatory region of cTnI and the regulatory domain of cTnC, thus decreasing FRET distances between cTnC and cTnI ( and ). This phosphorylation-induced enhancement of the cTnC-cTnI interaction may play a role in regulating the thin filament from the “blocked” to “closed” states. Since these structural changes in thin filaments are similar to those induced by strong crossbridges, our data also suggests that the “closed” to “open” states may also be enhanced by pseudo-phosphorylation of cTnI by relieving the inhibition of Tm movement on actin surface.
A consequence of the enhanced cTnC-cTnI interaction, caused by pseudo-phosphorylation of cTnI(S151E), is an increased sensitivity of cardiac thin filaments to Ca2+. For example, S151E mutation increased Ca2+ sensitivity of structural transition between cTnI and cTnC () as monitored by FRET distances between the central helix of cTnC and residues 160 and 167 of cTnI. Further additional increase in Ca2+ sensitivity was observed when strongly-bound S1 was present (). These additive effects of pseudo-phosphorylation of cTnI and strongly-bound S1 on Ca2+ sensitivity suggest a complex nature of Ca2+ binding to cardiac thin filaments. It is possible that Ca2+ sensitivity of thin filaments may be determined by structural dynamics associated with the whole C-domain of cTnI rather the regulatory region alone. It is likely that the phosphorylation-induced change is attributed to the enhanced interaction between the regulatory region of cTnI and cTnC caused by the charge modification. The additional structural change caused by strongly-bound S1, however, may be contributed to by other functional regions of cTnI, such as the inhibitory region and the mobile domain. These two regions are known as actin-binding regions, and their structural dynamics are sensitive to the presence of strong crossbridges.
In our Ca2+
titration experiments, we noticed that the values of pCa50
and the Hill coefficient obtained from FRET titrations of reconstituted thin filament samples () were different from the values obtained from pCa-force measurements with the reconstituted muscle fiber bundles (). These discrepancies between the two systems could be caused by different reasons. First, the force measurements of muscle fiber bundles were performed under isometric conditions at room temperature (20°C). FRET measurements were carried out in reconstituted thin filaments at 10°C. Second, FRET titrations were performed in reconstituted thin filaments that lack an ordered protein lattice structure present in sarcomeres of muscle fibers. The lattice structure in intact preparations provides optimal geometric and mechanical constraints on protein-protein interactions, which are likely to have an impact on the overall structural dynamics. The absence of protein lattice structure may also affect the cooperativity (the Hill coefficient) of the reconstituted system. For example, low values of the Hill coefficient are typically observed in the reconstituted samples1; 9; 11
. However, in a recent FRET measurement in muscle fiber bundles, we found that Ca2+
sensitivity and the Hill coefficient associated with the opening of the N-domain in cTnC were comparable to values obtained from simultaneous measurement of force and ATPase activity in normal muscle fibers (results will be presented in a new manuscript). Therefore, our results provide evidence for the importance of temperature and protein lattice structure in determining Ca2+
sensitivity and cooperativity of the thin filament.
In previous study with reconstituted samples, we observed that the presence of strongly-bound S1 decreased the Hill coefficients12
. This decrease is likely due to the disengagement of the inhibitory region of cTnI from actin when Tm shifts its position from the “closed” state to the “open” state. However, in this study the Hill coefficient was not significantly affected by the presence of either the strongly-bound S1 or the phosphorylation mimic of cTnI. It is unlikely that theFRET probes used in this study are limiting the cooperativity in the cTnC-cTnI interaction since these labeled proteins have no detrimental effect on maximal force, ATPase activity and Ca2+
sensitivity of skinned muscle fibers. It is possible that the cooperativity of the system monitored by FRET distances between the central helix of cTnC and the regulatory region of cTnI is insensitive to the presence of the strongly-bound S1 and phosphorylation mimic of cTnI. However, this speculation requires further investigation.
-mediated effects on cTnC-cTnI interactions play central roles in activating and deactivating muscle contraction by coupling Ca2+
-induced changes in cTnC to the attachment and detachment of strong crossbridges to and from actin. Our previous study suggested that Ca2+
-induced interactions between cTnC and the regulatory region of cTnI could be kinetically coupled with the processes that regulate cTnI-actin interactions9
. Consistent with our previous report 9
, our stopped-flow measurements showed fast kinetics of structural changes associated with the separation of the regulatory region of cTnI from cTnC upon Ca2+
removal. Thus, it is likely that this fast structural transition is kinetically coupled with the Ca2+
-induced closing of the regulatory N-domain of cTnC and the dynamic interaction between the mobile domain of cTnI and actin to initiate the movement of Tm on the surface of actin filament that regulates crossbridge detachment. However, our data showed that the presence of strong crossbridges had significant feedback impact on these structural transitions. For example, the kinetics of the Ca2+
dissociation-induced interaction between cTnC and the regulatory region of cTnI decreased by 28% – 64% in the presence of strongly-bound S1 (). This is because the strong crossbridges effectively hinder the movement of Tm from the “open” to the “closed” position on the actin surface and slow the kinetics of structural changes that switch cTnI from interacting with cTnC to interacting with actin upon Ca2+
removal. When the pseudo-phosphorylation of cTnI (S151E) was present, Ca2+
dissociation-induced kinetics decreased to the level observed in the presence of strongly-bound S1, even though no strongly-bound S1 was present. No significant additional reduction in the structural kinetics was observed when strongly-bound S1 was introduced. These results suggest that both the pseudo-phosphorylation cTnI (S151E) and strongly-bound S1 have similar effects on Ca2+
dissociation-induced structural interactions between the regulatory region of cTnI and cTnC, but with different mechanism.
To further understand the mechanism of PAK3 phosphorylation of cTnI on thin filament regulation, we measured Ca2+-activated maximal tension and ATPase activity of cardiac muscle fiber bundles () reconstituted with the PAK3 phosphorylation mimic of cTnI(S151E). cTnI(S151E) induced a statistically significant decrease in maximum ATPase activity, but only a marginal decrease in the maximum tension (). These changes in maximal tension and ATPase activity accounted for a marginal change in the tension cost, which did not provide any meaningful linkage between the pseudo-phosphorylation-induced slow kinetics observed in the Ca2+ dissociation-induced thin filament relaxation and crossbridge detachment kinetics.
In summary, we present novel information regarding structural and kinetic effects of PAK3 phosphorylation of cTnI at residue Ser151 on the Ca2+-induced thin filament regulation of cardiac muscle function. The enhanced cTnC-cTnI interaction caused by charge modification within the regulatory region of cTnI provides a molecular basis for the PAK3 phosphorylation-induced changes in structure, Ca2+ sensitivity, and relaxation kinetics of the thin filament. Our new findings shed light on the molecular basis of how the Ca2+-activated cardiac thin filament regulation is modulated by strongly-bound crossbridges and PAK3 phosphorylation of cTnI.