Mechanism of Zn²⁺ and Ca²⁺ Binding to Human S100A1

S100A1 is a member of the S100 family of small ubiquitous Ca²⁺-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn²⁺, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (K_D~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca²⁺ and Zn²⁺ and QM/MM molecular modeling we conclude that Zn²⁺ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn²⁺ even when the EF-hands are saturated by Ca²⁺, resulting in a 2Ca²⁺:S100A1:2Zn²⁺ conformer. Finally, we show that, in contrast to calcium, an excess of Zn²⁺ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca²⁺ (K_D~0.16 and 24 μm) than was previously reported for S100A1, which would allow this protein to function as a Ca²⁺/Zn²⁺-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.