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Abstract
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 (KD~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+ (KD~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.
Highlights
In this study, using electrospray mass-spectrometry, isothermal titration calorimetry (ITC), dynamic light scattering (DLS), differential scanning fluorimetry, and Quantum mechanics/molecular mechanics (QM/MM) molecular modeling, we investigated the mechanisms of Ca2+ and Zn2+ coordination in S100A1, including the structure and interrelations of its metal-binding sites
The stoichiometry of Ca2+ and Zn2+ binding to S100A1 was first analyzed by electrospray ionization mass-spectrometry (ESI-MS), as this method allows the direct monitoring of complexes of proteins with different metals
In the presence of calcium or zinc alone, S100A1 existed as five species corresponding to apo-protein and its conformers with one, two, three or four bound cations, the content of which decreases in the order 1Me2+ -S100A1 > 2Me2+ -S100A1
Summary
The S100 protein family encompasses small (~10 kDa) ubiquitous two-EF-hand Ca2+ binding proteins, normally existing as homo- or heterodimers and acting as both secreted and intracellular signaling molecules [1]. When released from cells, they display cytokine-like activity and bind to cell surface receptors (like toll-like receptors and G-proten-coupled receptors) or interact with growth. Biomolecules 2021, 11, 1823 factors, thereby regulating the activity of different immune cells, chondrocytes, myoblasts, cardiomyocytes, epitheliocytes, astrocytes, glial cells and neurons (reviewed in [2]). Altered expression of brain-specific S100 proteins is associated with decreased motor and cognitive skills and neurological damage characteristic of Alzheimer’s disease (AD) and Parkinson’s disease, and several members of the S100 family are found in the amyloid plaques and cerebrospinal fluid of AD patients [4,5,6,7]. Despite extensive knowledge of the cellular and tissue functions of S100 proteins, understanding of the exact mechanisms underlying their signaling activity remains scarce, especially regarding Ca2+ -sensitivity and interrelations between multiple isoforms of these proteins
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