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 of Cu(II) and Cu(I) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau (Fig. 1). R1 (HL): Ac-257VKSKIGSTENLKHQGGG273-NH2 R3 (L): Ac-323GSLGNIHHKPGGG335-NH2 Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where the imidazole ring, two amidic nitrogen atoms, and a water molecule occupy the equatorial coordination positions of copper(II). As for the R3 peptide, at pH 7.4 [CuL]2+ and [CuLH-1]+ are the two most abundant species (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. Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where the imidazole ring, two amidic nitrogen atoms, and a water molecule occupy the equatorial coordination positions of copper(II). As for the R3 peptide, at pH 7.4 [CuL]2+ and [CuLH-1]+ are the two most abundant species (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. Copper(I) adducts with the R1 and R3 tau fragments were investigated via spectrophotometric competition titrations with the metallochromic ligand ferrozine (Fz).[4] The chromophoric complex [CuI(Fz)2]3- (having two characteristic absorption bands at 470 nm and 600 nm) is formed by titrating a Cu(I) solution with ferrozine. The back titration of this solution with the R1 and R3 fragments, led to a decrease in the absorbance values (Fig. 2). A significative change in the absorbance, which decreases of almost 0.40 units, is observed upon the addition of R3 to [CuI(Fz)2]3-. On the contrary, in the case of the R1 peptide, the absorbance decrease of only 0.20 units can be fully accounted by dilution effects. Data treatment using HypSpect program yields a log β value of 10.1(2) for the Cu(I)-R3 complex, while for the back titration of [CuI(Fz)2]3- with R1 it 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 redox behavior of these complexes will be discussed in terms of their speciation. Also, an insight of the role of the copper adducts with R1 and R3 in catecholase activity will be given. 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. [3] Zweckstetter, Biochemistry 2008, 47, 10841-51. [4] Z. Xiao, L. Gottschlich, R. Meulen, S. R. Udagedara, A. G. Wedd, Metallomics, 2013, 5, 501-513. ACKNOWLEDGEMENTS The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW).

Copper binding to R1 and R2 fragments of Tau protein

Denise Bellotti;Maurizio Remelli;
2019

Abstract

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 of Cu(II) and Cu(I) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau (Fig. 1). R1 (HL): Ac-257VKSKIGSTENLKHQGGG273-NH2 R3 (L): Ac-323GSLGNIHHKPGGG335-NH2 Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where the imidazole ring, two amidic nitrogen atoms, and a water molecule occupy the equatorial coordination positions of copper(II). As for the R3 peptide, at pH 7.4 [CuL]2+ and [CuLH-1]+ are the two most abundant species (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. Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where the imidazole ring, two amidic nitrogen atoms, and a water molecule occupy the equatorial coordination positions of copper(II). As for the R3 peptide, at pH 7.4 [CuL]2+ and [CuLH-1]+ are the two most abundant species (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. Copper(I) adducts with the R1 and R3 tau fragments were investigated via spectrophotometric competition titrations with the metallochromic ligand ferrozine (Fz).[4] The chromophoric complex [CuI(Fz)2]3- (having two characteristic absorption bands at 470 nm and 600 nm) is formed by titrating a Cu(I) solution with ferrozine. The back titration of this solution with the R1 and R3 fragments, led to a decrease in the absorbance values (Fig. 2). A significative change in the absorbance, which decreases of almost 0.40 units, is observed upon the addition of R3 to [CuI(Fz)2]3-. On the contrary, in the case of the R1 peptide, the absorbance decrease of only 0.20 units can be fully accounted by dilution effects. Data treatment using HypSpect program yields a log β value of 10.1(2) for the Cu(I)-R3 complex, while for the back titration of [CuI(Fz)2]3- with R1 it 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 redox behavior of these complexes will be discussed in terms of their speciation. Also, an insight of the role of the copper adducts with R1 and R3 in catecholase activity will be given. 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. [3] Zweckstetter, Biochemistry 2008, 47, 10841-51. [4] Z. Xiao, L. Gottschlich, R. Meulen, S. R. Udagedara, A. G. Wedd, Metallomics, 2013, 5, 501-513. ACKNOWLEDGEMENTS The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2479807
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