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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.

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation., (Copyright © 2018. Published by Elsevier Ltd.)

Tau is a naturally disordered microtubule associated protein which forms intraneuronal aggregates in several neurodegenerative diseases including Alzheimer's disease (AD). It was reported that zinc interaction with tau protein can trigger its aggregation. Recently we identified three zinc binding sites located in the N-terminal part, repeat region and the C-terminal part of tau. Here we characterized zinc binding to each of the three sites using isothermal titration calorimetry (ITC) and determined the impact of each site on aggregation using dynamic light scattering (DLS) assays. First, we confirmed the presence of three zinc binding sites on tau and determined the thermodynamic parameters of binding of zinc to these sites. We found a high-affinity zinc binding site located in the repeat region of tau and two N- and C-terminus binding sites with a lower binding constant for zinc. Second, we showed that tau aggregation necessitates zinc binding to the high affinity site in the R2R3 region, while LLPS necessitates zinc binding to any two binding sites. With regard to the role of zinc ions in the aggregation of proteins in neurodegenerative diseases, these findings bring new insights to the understanding of the aggregation mechanism of tau protein induced by zinc. • Zinc is known to trigger tau aggregation and droplet formation. • Three zinc-binding sites have been identified in a NMR study. • Zinc binding site located in the R2R3 domain is crucial for tau aggregation. • Zinc binding to only one site does not induce tau aggregation nor LLPS. • Any two of three existing zinc-binding sites are necessary to induce droplets. [ABSTRACT FROM AUTHOR]