Mechanism of Zn(2+) and Ca(2+) Binding to Human S100A1

S100A1 is a member of the S100 family of small ubiquitous Ca(2+)-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 Zn(2+), 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 Ca(2+) and Zn(2+) and QM/MM molecular modeling we conclude that Zn(2+) high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn(2+) even when the EF-hands are saturated by Ca(2+), resulting in a 2Ca(2+):S100A1:2Zn(2+) conformer. Finally, we show that, in contrast to calcium, an excess of Zn(2+) produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca(2+) (K(D)~0.16 and 24 μm) than was previously reported for S100A1, which would allow this protein to function as a Ca(2+)/Zn(2+)-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.