Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations

Adrenaline acts on β1 receptors in the heart muscle to enhance contractility, increase the heart rate, and increase the rate of relaxation (lusitropy) via activation of the cyclic AMP-dependent protein kinase, PKA. Phosphorylation of serines 22 and 23 in the N-terminal peptide of cardiac troponin I is responsible for lusitropy. Mutations associated with cardiomyopathy suppress the phosphorylation-dependent change. Key parts of troponin responsible for this modulatory system are disordered and cannot be resolved by conventional structural approaches. We performed all-atom molecular dynamics simulations (5 × 1.5 μs runs) of the troponin core (419 amino acids) in the presence of Ca2+ in the bisphosphorylated and unphosphorylated states for both wild-type troponin and the troponin C (cTnC) G159D mutant. PKA phosphorylation affects troponin dynamics. There is significant rigidification of the structure involving rearrangement of the cTnI(1–33)–cTnC interaction and changes in the distribution of the cTnC helix A/B angle, troponin I (cTnI) switch peptide (149–164) docking, and the angle between the regulatory head and ITC arm domains. The familial dilated cardiomyopathy cTnC G159D mutation whose Ca2+ sensitivity is not modulated by cTnI phosphorylation exhibits a structure inherently more rigid than the wild type, with phosphorylation reversing the direction of all metrics relative to the wild type.


Phosphorylation-dependent interactions of NcTnI:
The major interaction between NcTnC and unphosphorylated NcTnI is TnC Asp33, in the EF hand loop I, with TnI Arg 19,20 and 21 this is significant as Asp 33 is one of the few cardiac specific variants in cTnC (Gly33 in skeletal muscle TnC) (Figure 2C).Upon phosphorylation, there is a cumulative loss of these interactions.This is accompanied by altered NcTnC-NcTnI interactions increasing the NcTnC Asp25 to NcTnI Arg12 interaction and the Glu32 to Arg12, both TnC residues being at the end of the A helix Interactions of Ser 22 and 23 with TnC Lys39 are formed upon phosphorylation.
In the unphosphorylated state there is no ionic interaction of the Ser 22 and 23 with TnC Lys39, upon phosphorylation the interaction with Ser 22 and 23 increases significantly to ~80% (green circle).The resulting shift of NcTnI-NcTnC interactions changes the location of NcTnI on NcTnC and results in Lys39 in the B helix being pulled towards the phosphorylated serines of NcTnI, confirmed by the formation of an interaction between TnC Thr38 and TnI.As a consequence, there is also a very noticeable change in the orientation of the end of the B-Helix relative to the A helix.There is a loss of interaction of NcTnC Glu40 with NcTnI Arg20/21.Uniquely, there is a strong phosphorylation effect on the interaction of the Phenylalanine residues in the A and D helices.Whilst the interaction between the end of the A helix Phe 24 with the start of the D helix Phe 74 and 77 is relatively constant, the very end of the A helix Phe27 increases its interaction upon phosphorylation with the D helix Phe77.

Phosphorylation-dependent interactions of cTnI switch peptide:
The troponin I switch peptide, 149-164, is docked onto the hydrophobic patch of NcTnC formed by helix A and B. It is anchored to NcTnC by a stable interaction between NcTnC Glu19 and cTnI Arg161, but the repositioning of cTnC helix B upon phosphorylation alters the hydrophobic patch structure and results in repositioning of the switch peptide.There is a noticeable loss of interactions between TnC helix B and the C-terminal part of the switch

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peptide.The interaction of NcTnC Lys43 with the switch peptide residues Glu164 and Arg169 decreases and the interaction of cTnC Arg 46 with ALA170 also decreases.At the N-terminal end of the switch peptide there is increased hydrogen bonding between cTnC Cys84 and cTnI Ala150 and cTnC Ser89 and Gly91 with cTnI Gly159.
Phosphorylation-dependent interactions of cTnI 'inhibitory' peptide: Repositioning of the TnI switch peptide relative to cTnC helices A and B on phosphorylation alters the adjacent 'inhibitory peptide', TnI 136-148.The cTnC C-helix moves with the B helix, thus altering its interactions with the middle of the 'inhibitory peptide.The arginine's in this peptide (140,144,145,147,149) form ionic interactions with cTnC, notably at the start of Helix C and the end of Helix D that shift on phosphorylation.cTnC Asp62 and Glu63 increase their interactions with cTnI Arg140 and 145; TnC Asp87 also increases its interaction with cTnI Arg 145 and 147.cTnC Glu56 loses interaction with cTnI Arg144 and 145, however, there is and increased interaction with cTnI Val146.cTnC Glu59 also losses its ionic interaction with cTnI Arg140 and 145.Overall, upon phosphorylation, the inhibitory peptide repositions towards the start of C-helix.

Intra NcTnI interactions
The binary nature of the increase in interactions between residues of TnC and TnI described above are also echoed by new intra-NcTnI interactions formed between the phosphate groups of Ser 22 and 23 with TnI Arg 19,21 and 26.There is a very clear preference for the Ser 23 to interact with TnI Arg 19,21 and 26, from 0% in the unphosphorylated case.Only Arg 19 makes significant contact with phosphorylated Ser 22 and Arg 20 only interacts weakly in the phosphorylated state

Intra cTnC interactions
Rearrangement of TnC helix B on phosphorylation alters helices C and D. On phosphorylation, contact between the end of D-helix Arg83 and residues Asp88 and Glu155 is weakened, with the linker moving away from the D helix.At the same time, the linker region, Asp88 and Gly91, move collectively towards C-TnC, accompanied by changes in the E-Helix.At the start of E-helix Ser93 is moving away from the rest of the helix.This is reflected in the reduction of its interactions with Glu95, Glu96 and Leu97.The overall effect is that the TnC linker peptide lengthens upon phosphorylation increasing the distance between the NcTnC and CcTnC domains

cTnC-CcTnT interactions
There is a medium interaction formed between the NcTnC EF1 hand, Asp 33, and the CcTnT Arg 286, upon phosphorylation which correlates with the repositioning of the cTnC Lys 39 and the phosphorylated serines described previously.Interactions between the EF3 hand of the CcTnC and the CcTnT are significantly modulated by phosphorylation.The interaction between CcTnC Asp 105 and TnT Tyr 259 increases significantly, whilst CcTnC

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Asp 109 with CcTnT Asn 262 and Asn 266 also increase.CcTnC residues at the end of the EF3 hand Gly 110 and Tyr 111 also experience significant increases in interactions with CcTnT residues Asn 266 and Asp 270.Further phosphorylation induced changes between CcTnC and CcTnT are also observed in the region of the EF4 hand and helix H. Specifically the interaction between CcTnC Arg 147 and CcTnT Asp 270 increases and that of Asp 151 with Arg 267.

cTnI-CcTnT interactions
There is formation of an interaction between the CcTnT Lys280 and the phosphorylated Ser 22 and 23 which is completely absent in the unphosphorylated state.There is also an increased cumulative interaction between TnI Arg 19,20 and 21 and CcTnT Lys280 upon phosphorylation which like the interaction with the Ser 22 and 23 is absent in the unphosphorylated state

Phosphorylation-independent changes
The most significant change from the WT is the formation of a strong, ~80%, ionic interaction between Arg 83, at the bottom of the D-helix and the Asp159, the end of the H-helix (green circle) -Supp 6G.Arg 83 also forms a strong interaction with the N-helix of cTnC which is phosphorylation dependent in the WT at the expense of the internal interaction between Glu155 and Lys 158 (pink circle).The aromatic interactions between the end of A-helix Phe's 24 and 27 with the Phe's 74 and 77 at the start of the D-helix are phosphorylation independent and broadly of the same magnitude as the WT, though the WT exhibits phosphorylation dependence in respect of the Phe 27 interaction with Phe77.

Phosphorylation-dependent changes
Structural differences accompanying phosphorylation of G159D, can be observed in respect of the interaction of the N-helix of cTnC and the beginning of the H1 helix of the NcTnI, specifically the interaction of Glu 10 and Ser 41 and 43, in WT the interactions increase whilst in the G159D they decrease.These differences are further amplified when examining the differences in the anchoring of the NcTnI to the A-helix of NcTnC, the interactions of cTnC Glu15 and 19 with cTnI Arg 9 in WT are constant, whilst for G159D they decrease significantly.These indicate a large loss of anchoring of the NcTnI, however, there is an accompanying increase in the binding of the CcTnI switch peptide to the TnC Glu19 whilst in the WT this is relatively static.
Addressing the self-interactions of cTnI in the G159D mutant in general it is observed that the changes upon phosphorylation mirror those observed in the WT, though often the magnitude of the change is larger than that observed for the WT.This is observed in S30 particular for the interaction of residues Arg 19 through Ala 27, for example Arg19 with Ser 22 and 23 in the G159D increases from 0% in both cases to ~45%, compared with an asymmetric 23% and 68 in the WT; though the cumulative change is the same.For the interaction of TnI Arg 20 and Arg 21 with Ser 22 and Ser 23 respectively we observe the largest changes in magnitude for G159D with respect to WT.This clearly illustrates that phosphorylation has a more pronounced effect on the local structure in this region of the complex.
Phosphorylation-dependent changes in G159D that are the reverse of WT TnC Asp25 and Glu32 with TnI Arg12 the interaction in the G159D decreases from whilst in the WT they increase (orange circle 6H).In the linker region cTnC-cTnC interactions(supp 6I) for residues Lys 90 with Glu 94 (blue circle) and Asp159 (green circle) interactions increase upon phosphorylation in G159D whilst for WT they decrease.WT interactions between Ser93 and Glu 96 and Leu 97 decreased substantially upon phosphorylation, but for G159D these increase; this is also observed in the interaction of Glu 94 with Ser 98 which remains constant at 90% whilst in the WT there is a decrease.
CcTnC Ala 108 interaction with CcTnT Asn266 increases from 0% to ~20% in contrast to WT where this is essentially completely lost upon phosphorylation.Interactions of Asp109 with CcTnT Asn 262 and 266 decrease by the same magnitudebut increase in WT.For CcTnC Gly110 with CcTnT Asn 266 there is a small decrease whilst in the WT there is a large increase.The largest difference between the G159D and the WT is for CcTnC Tyr111 interacting with CcTnT Asp 270: in G159Dthis decreases by ~20% whilst for the WT it increases by ~20% on phosphorylation.
In the EF4 hand of G159D CcTnC Asn 143 and 144 interactions with the CcTnT Lys 282 are formed on phosphorylation where there is a complete absence of interaction in the WT both states.There are also a number of more subtle differences between the interaction of the CcTnT and cTnI in G159D compared to the WT, in particular CcTnT Gly 279 with TnI Lys 139 and 140, in WT there is an increase upon phosphorylation whilst in G159D there is no interaction in either state.A similar pattern is seen for CcTnT Lys 280 with NcTnI Arg 19 and 21, in WT these increase upon phosphorylation, whereas in G159D there is no interaction in either state.