Disease Mechanisms DOI: 10.1002/ange.201005838 Copper(II) Coordination to Amyloid b : Murine versus Human Peptide** HØlne Eury, Christian Bijani, Peter Faller, and Christelle Hureau* In Alzheimers disease (AD), the amyloid b (Ab) peptide seems to play a causative role. Ab is the major constituent of amyloid plaques, a hallmark of AD. According to the amyloid cascade hypothesis, in AD, the aggregation of Ab leads to the formation of toxic species, which induce neuronal cell death. It has been proposed that reactive oxygen species (ROS) are produced, and that these species mediate cell toxicity. [1, 2] Although still under debate, [3] a large body of evidence suggests that metallic ions (copper, zinc, and iron) play a role in the etiology of AD. [3–6] For example, amyloid plaques extracted from human brains contain high amounts of Cu II and Zn II ions [7] bound to the Ab peptide. [8, 9] Chelators were able to partially solubilize the plaques, [8] and studies on neuronal cell culture and transgenic mice supported the involvement of ions in Ab metabolism. [10, 11] Copper(II) can be released in the synaptic cleft and can reach concentrations up to 15 mm. [5] This value is in line with the possibility of Cu II binding to Ab in vivo, since a dissociation constant in the picomolar range has been determined for the Cu II –Ab species. [12] Furthermore, in vitro aggregation of the Ab peptide can be modulated by Cu and Zn ions, [12–14] and because of its redox nature, Cu may play a role in ROS production. [15] These observations and hypotheses explain the intensive research on the modulation of metal-ion homeo- stasis as a therapeutic approach. [3, 16] A better understanding of the AD mechanisms requires investigations on mouse and rat animal models. [17, 18] However, these animals, whose peptide differs from the human Ab peptide by three point mutations, do not show amyloid deposition. [17, 18] Consequently, studies are performed on transgenic mice or rats that produce the human Ab (hAb) peptide in addition to their own peptide (mAb). Cu II coordination to murine and human peptides has been proposed to differ. [19–21] Thus, in the present study, to explain the distinct Cu II coordination to hAb and mAb, we used complementary spectroscopic techniques to determine the crucial mutation(s). We also propose Cu II –mAb structural models. Finally, we discuss possible consequences of such differences in Cu II coordination with respect to the use of mice or rats as AD animal models. We examined the coordination of Cu II to six peptides: hAb (DAEFRHDSGYEVHHQK; see Scheme S1 in the Supporting Information), Y10F-hAb, H13R-hAb, R5G- hAb, mAb (DAEFGHDSGFEVRHQK; see Scheme S2 in the Supporting Information), and F10Y-mAb (or R5G-H13R- hAb). These shorter 16-residue peptides were used as valuable models of Cu II binding to the full-length pep- tides. [22–24] Indeed, no differences in spectroscopic signature, [22] binding affinity, [24] or ROS production [23] have been observed between the truncated and full-length hAb peptides. Two peptide families can be distinguished from the spectroscopic signatures of their Cu II complexes (see Figures S1–S5 and Table S1 in the Supporting Information): hAb, Y10F-hAb, and H13R-hAb (humanlike family), and mAb, F10Y-mAb, and R5G-hAb (murine-like family). Thus, the key mutation between the hAb and mAb peptides with regard to Cu II binding is the R5G mutation. For both families, two Cu II complexes that differ in the protonation state of the peptide are present near the physiological pH value, namely, compo- nents I and II. Figure 1 shows the differences between the CD and EPR spectroscopic signatures of Cu II –hAb and Cu II –mAb solutions at pH 6.7 and 5.4, at which I is predominant, and at pH 8.7 and 7.6, at which II is predominant. The pKa(I/II) values are close to pH 7.7 for Cu II complexes of the human- like peptides and close to pH 6.2 for the murine-like family (see Figures S3 and S5 and Table S1 in the Supporting Information). We previously described copper(II)-induced modification of the peptide NMR spectroscopic signature to determine the Cu II -binding sites of hAb. [25] The results obtained were in line with most previous studies [12, 19, 22, 26, 27] and showed that the equatorial binding site of component I is formed by the NH 2 group of Asp1, two of the three imidazole rings of His6, His13, and His14, and a CO function. At higher pH values, deprotonation of the Asp1 ÀAla2 peptide bond leads to the replacement of one imidazole ring with the Asp1 ÀAla2 deprotonated amide (amidyl) ligand. In this study, we used NMR spectroscopy to gain more insight into Cu II coordination to the mAb peptide. We recorded 1 H, 13 C, and 2D NMR spectra of mAb peptide at pH 5.4 and 7.6 with or without a substoichiometric amount of Cu II ions (see Figures S6–S14 in the Supporting Information). At pH 5.4 and in the presence of Cu II , the side chains of Asp, [*] H. Eury, Dr. C. Bijani, Prof.Dr. P. Faller, Dr. C. Hureau CNRS, LCC (Laboratoire de Chimie de Coordination) 205, route de Narbonne, 31077 Toulouse (France) and UniversitØ de Toulouse, UPS, INPT, LCC 31077 Toulouse (France) Fax: (+ 33) 5-6155-3003 E-mail: christelle.hureau@lcc-toulouse.fr [**] This research was supported by a grant from the Agence Nationale de la Recherche, Programme Blanc NT09-488591, “NEUROME- TALS” (P.F. and C.H.). C.H. acknowledges Dr. I. Sasaki, Dr. P. Dorlet, and Dr. L. Sabater for valuable comments on the manu- script, L. Rechignat for EPR measurements, and Y. Coppel and C. L. Serpentini for their help with NMR and CD experiments, respec- tively. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201005838. Angewandte Chemie 931 Angew. Chem. 2011, 123, 931 –935  2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim