Desensitization of the Cardiac Troponin Complex by TnI Phosphorylation and Epigallocatechin-3-Gallate.

Cardiac contractility is regulated by the Ca2+ sensitivity of thin filaments, largely controlled by troponin I (TnI). Phosphorylation of TnI at Ser23/24 and the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) both reduce thin filament Ca2+ responsiveness, yet the underlying structural mechanisms remain incompletely defined. Here, we integrate in vitro motility assays with AlphaFold 3 modeling and molecular dynamics (MD) simulations to characterize the effects of TnI phosphorylation and EGCG on the troponin complex. Motility assays using reconstituted thin filaments showed that TnI Ser23/24 phosphorylation reduced maximum sliding velocity (V max) by -49 ± 7% and shifted pCa50 by -3 ± 2%. EGCG caused a greater decrease in V max (-58 ± 8%) and a larger pCa50 shift (-8 ± 4%), consistent with desensitization to Ca2+ in both cases. Structural models generated via AlphaFold 3 predict that Ser23/24 phosphorylation induces an α-helical conformation that repositions the TnI N-terminal extension away from the Troponin C (TnC) N-lobe. On/off time analyses from MD simulations showed rapid transitions (∼0.18 ps on, ∼0.16 ps off), consistent with transient, functionally meaningful interactions at the TnI-TnC interface of unphosphorylated TnI. Docking simulations identified a probable EGCG binding site at the interface between the TnC C-lobe (residues 120-161) and TnI's IT-arm and upstream unstructured region (residues 34-71), stabilized by hydrogen bonds to both subunits. MD simulations revealed recurrent, short-lived hydrogen bonding between TnI and TnC. Together, these findings support an allosteric desensitization model where phosphorylation modulates the TnI-TnC N-lobe interaction, while EGCG binding could modify conformational changes at the TnC C-lobe and contigous TnI domains. These insights may guide small-molecule design to modulate Ca2+ sensitivity in cardiac disease.