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 (Vmax) by −49 ± 7% and shifted pCa50 by −3 ± 2%. EGCG caused a greater decrease in Vmax (−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.