Molecular Basis of S100A1 Activation at Saturating and Subsaturating Calcium Concentrations

The S100A1 protein mediates a wide variety of physiological processes through its binding of calcium (Ca²⁺) and endogenous target proteins. S100A1 presents two Ca²⁺-binding domains: a high-affinity “canonical” EF (cEF) hand and a low-affinity “pseudo” EF (pEF) hand. Accumulating evidence suggests that both Ca²⁺-binding sites must be saturated to stabilize an open state conducive to peptide recognition, yet the pEF hand’s low affinity limits Ca²⁺ binding at normal physiological concentrations. To understand the molecular basis of Ca²⁺ binding and open-state stabilization, we performed 100 ns molecular dynamics simulations of S100A1 in the apo/holo (Ca²⁺-free/bound) states and a half-saturated state, for which only the cEF sites are Ca²⁺-bound. Our simulations indicate that the pattern of oxygen coordination about Ca²⁺ in the cEF relative to the pEF site contributes to the former’s higher affinity, whereas Ca²⁺ binding strongly reshapes the protein’s conformational dynamics by disrupting β-sheet coupling between EF hands. Moreover, modeling of the half-saturated configuration suggests that the open state is unstable and reverts toward a closed state in the absence of the pEF Ca²⁺ ion. These findings indicate that Ca²⁺ binding at the cEF site alone is insufficient to stabilize opening; thus, posttranslational modification of the protein may be required for target peptide binding at subsaturating intracellular Ca²⁺ levels.