Copper binding to R1 and R3 fragments of Tau protein

Tau is a 441-mer peptide present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble amyloid aggregates of tau are associated with over 20 neurological disorders known as tauopathies, among which is Parkinson.[1] In neurons, tau binds tubulin through its microtubule binding domain which comprises four repeats (R1- R4) characterized by the presence of histidine residues. These regions are potential binding sites for metal ions.[2] The elucidation of the binding capacities toward metal ions, especially those redox active such as copper(II), may shed light on the biomolecular processes that underlie the progression of tauopathies.[3] In this contribution we examine the stability and the structural models of Cu(II) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau (Fig. 1). Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where R1 is tridentate: the copper(II) equatorial coordination positions are occupied by the imidazole ring, two amidic nitrogen atoms, and a water molecule. As for the R3 peptide, at pH 7.4 the two most abundant species are [CuL]2+ and [CuLH-1]+ (in a ratio of ca. 1:2). In the case of [CuL]2+, the two imidazole groups of R3 and one deprotonated amidic nitrogen atom are bound to the equatorial plane. In [CuLH-1]+, a further amidic nitrogen bounds the metal ion in the equatorial plane, most likely pushing one imidazole group to the axial position. The redox and NMR behavior of these complexes will be discussed in terms of their speciation. Having the Cu(II)-tau peptides binding constants in our hands, we decided to investigate the copper(I) adducts with the R1 and R3 tau fragments via spectrophotometric competition titrations with the metallochromic ligand ferrozine (Fz).[4] The titration of a Cu(I) solution with ferrozine causes the formation of the chromophoric complex [CuI(Fz)2]3- which presents two characteristic absorption bands at 470 nm and 600 nm. By back titrating this solution with the R1 and R3 fragments, we observed a decrease in the absorbance values (Fig. 2). The addition of R3 to [CuI(Fz)2]3- causes a significative change in the absorbance, which decreases of almost 0.40 units. As for the R1 peptide, a decrease of only 0.20 units is observed and can be fully accounted by dilution effects. Data treatment using HypSpect program yields a log β value of 5.9(1) for the Cu(I)-R3 complex. Data treatment for the back titration of [CuI(Fz)2]3- with R1 confirms the absence of significant interactions of Cu(I) with R1. NMR data suggest that the binding of Cu(I) to R3 occurs at the tandem HH site, as it occurs for Cu(II). The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW). References: [1] M. Goedert, D. S. Eisenberg, R. A. Crowther, Annu. Rev. Neurosci. 2017, 40, 189-210. [2] M. G. Savelieff, S. Lee, Y. Liu, M. H. Lim, ACS Chem. Biol., 2013, 8, 856-865. [3] A. Soragni, B. Zambelli, M. D. Mukrasch, J. Biernat, S. Jeganathan, C. Griesinger, S. Ciurli, E. Mandelkow, M. Zweckstetter, Biochemistry, 2008, 47, 10841-51. [4] B. Alies, B. Badei, P. Faller, C. Hureau, Chem. Eur. J., 2012, 18, 1161-1167.