Molecular Insights into the Calcium Binding in Troponin C Through a Molecular Dynamics Study

Calcium-binding proteins play critical roles in various biological processes such as signal transduction, cell growth, and transcription factor regulation. Ion binding and target binding of Ca2+-binding proteins are highly related. Therefore, understanding the ion binding mechanism will benefit the relevant inhibitor design towards the Ca2+ binding proteins. The EF-hand is the typical ion binding motif in Ca2+-binding proteins. Previous studies indicate that the ion binding affinity of the EF-hand increases with peptide length, but this mechanism has not been fully established. Herein, using molecular dynamics (MD) simulations, thermodynamic integration (TI) calculations, and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) analysis, we systematically investigated four Ca2+ binding peptides containing the EF-hand loop in the site III of rabbit skeletal troponin C (TnC). These four peptides have 13, 21, 26, and 34 residues, respectively. Our simulations reproduced the observed trend that the ion binding affinity increases with peptide length. Our results implied that the E-helix motif preceding the EF-hand loop, especially the Phe99 residue, plays a significant role in this regulation. The E-helix has a significant impact on the backbone and sidechain conformations of the Asp103 residue, rigidifying important hydrogen bonds in the EF-hand and decreasing the solvent exposure of the Ca2+ ion, hence leading to more favorable Ca2+ binding in longer peptides. The present study provides molecular insights into the ion binding in the EF-hand and establishes an important step towards elucidating the responses of Ca2+-binding proteins towards the ion and target availability.