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 Ca 2+ -binding proteins are highly related. Therefore, understanding the ion binding mechanism will benefit the relevant inhibitor design toward the Ca 2+ -binding proteins. The EF-hand is the typical ion binding motif in Ca 2+ -binding proteins. Previous studies indicate that the ion binding affinity of the EF-hand increases with the peptide length, but this mechanism has not been fully understood. Herein, using molecular dynamics simulations, thermodynamic integration calculations, and molecular mechanics Poisson-Boltzmann surface area analysis, we systematically investigated four Ca 2+ -binding peptides containing the EF-hand loop in site III of rabbit skeletal troponin C. These four peptides have 13, 21, 26, and 34 residues. Our simulations reproduced the observed trend that the ion binding affinity increases with the peptide length. Our results implied that the E-helix motif preceding the EF-hand loop, likely the Phe99 residue in particular, plays a significant role in this regulation. The E-helix has a significant impact on the backbone and side-chain conformations of the Asp103 residue, rigidifying important hydrogen bonds in the EF-hand and decreasing the solvent exposure of the Ca 2+ ion, hence leading to more favorable Ca 2+ binding in longer peptides. The present study provides molecular insights into the ion binding in the EF-hand and establishes an important step toward elucidating the responses of Ca 2+ -binding proteins toward the ion and target availability.