Identification of calcium binding sites on calsequestrin 1 and their implications for polymerization

Abstract

Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70–80 Ca²⁺ ions. However, the nature of Ca²⁺-binding sites has not yet been fully characterized. In this study, we employed in silico approaches to identify the Ca²⁺ binding sites and to understand the molecular basis of CASQ1–Ca²⁺ recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: 3UOM) and adding the missing C-terminal residues (aa350–364). Using this model we performed extensive 30 ns molecular dynamics simulations over a wide range of Ca²⁺ concentrations ([Ca²⁺]). Our results show that the Ca²⁺-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca²⁺-binding sites share a similar geometry and interestingly, the majority of them were found to be induced by increased [Ca²⁺]. We also found that the system shows maximal Ca²⁺-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data show that the CASQ1 back-to-back stacking is progressively stabilized by the emergence of an increasing number of hydrophobic interactions with increasing [Ca²⁺]. Further, this study shows that the CAS domain assumes a compact structure with an increase in Ca²⁺ binding, which suggests that the CAS domain might function as a Ca²⁺-sensor that may be a novel structural motif to sense metal. We propose the term “D_n-motif” for the CAS domain.