Zn(II) ions substantially perturb Cu(II) ion coordination in amyloid-β at physiological pH

Abstract

The interaction of Cu(II) and Zn(II) ions with amyloid-β (Aβ) plays an important role in the etiology of Alzheimer's disease. We describe the use of electron spin resonance (ESR) to measure metal-binding competition between Cu(II) and Zn(II) in amyloid-β at physiological pH. Continuous wave ESR measurements show that the affinity of Cu(II) toward Aβ(1-16) is significantly higher than that of Zn(II) at physiological pH. Importantly, of the two known Cu(II) coordination modes in Aβ, component I and component II, Zn(II) displaces Cu(II) only from component I. Our results indicate that at excess amounts of Zn(II) component II becomes the most dominant coordination mode. This observation is important as Aβ aggregates in the brain contain a high Zn(II) ion concentration. In order to determine details of the metal ion competition, electron spin echo envelope modulation experiments were carried out on Aβ variants that were systematically (15)N labeled. In the presence of Zn(II), most peptides use His 14 as an equatorial ligand to bind Cu(II) ions. Interestingly, Zn(II) ions completely substitute Cu(II) ions that are simultaneously coordinated to His 6 and His 13. Furthermore, in the presence of Zn(II), the proportion of Cu(II) ions that are simultaneously coordinated to His 13 and His 14 is increased. On the basis of our results we suggest that His 13 plays a critical role in modulating the morphology of Aβ aggregates.

Figure 1

Three proposed subcomponents for component I in Cu(II) coordinated to Aβ peptide. In subcomponent IA, His 6 and 13 simultaneously coordinate the Cu(II) ion in the uatorial plane. Subcomponent IB Cu(II) coordinates His 6 and 14, while subcomponent IC coordination contains His 13 and 14

Figure 2

CW-ESR spectra illustrating the reduction of Cu(II) intensity in the presence of Zn(II) when coordinated to Aβ(1–16) peptide at physiological pH. At equimolar amount, Zn(II) reduces the double integrated intensity of Cu(II) signal by ~ 25 % with respect to the no Zn(II) spectra, and at four equivalents of Zn(II) the signal intensity is reduced by ~ 40 %.

Figure 3

(a) Overlay of CW-ESR spectra of Aβ(1–16)-Cu(II) equimolar binary complex (black) and Aβ(1–16)-Cu(II)/Zn(II) equimolar ternary complex (grey) at pH 7.4 and (b) at pH 8.7. At pH 8.7 only the component II of Cu(II) binding is present. Interestingly double integrated intensity of the spectra remains almost the same, suggesting Zn(II) cannot compete with Cu(II) for component II coordination

Figure 4

Experimentally obtained and simulated three-pulse ESEEM spectra of the nonlabeled Aβ(1–16) peptide mixed with equimolar amounts of Cu(II) and Zn(II) at 2800 G at pH 7.4.

Figure 5

Three-pulse ESEEM spectra of the nonlabeled and single ¹⁵N labeled Aβ(1–16) variants mixed with equimolar amounts of Cu(II) and Zn(II) at 2800 G at pH 7.4 (peptide: Cu(II): Zn(II) =1:1:1). The decrease in intensity below 8 MHz in ¹⁵N labeled Aβ(1–16) variants gives the contribution of each histidine residue for component I in Aβ(1–16)-Cu(II)/Zn(II) complex. Aβ(1–16)H6[¹⁵N], Aβ(1–16)H13[¹⁵N], Aβ(1–16)H14[¹⁵N] denote peptides where His 6, His 13, and His 14 are labeled with ¹⁵N, respectively.

Figure 6

Three-pulse ESEEM spectra of the nonlabeled and double ¹⁵N labeled Aβ(1–16) variants mixed with equimolar amounts of Cu(II) and Zn(II) at 2800 G at pH 7.4(peptide: Cu(II): Zn(II) =1:1:1). Integrated area between 0 – 8 MHz gives the contribution of the nonlabeled histidine residue in double labeled Aβ(1–16) variants for component I in Aβ(1–16)-Cu(II)/Zn(II) complex. Aβ(1–16)H13,14[¹⁵N], Aβ(1–16)H6,14[¹⁵N], Aβ(1–16)H6,13[¹⁵N] denote peptides where His 13/14, His 6/14, and His 6/13 are labeled with ¹⁵N, respectively.

Figure 7

Overall population distribution of Cu(II) binding modes in Aβ(1–16) in the presence and the absence of Zn(II) at physiological pH. The proportion of Subcomponent IC, which may inhibit the formation of ordered fibrillar forms, is increased. Subcomponent IA is no longer present in the presence of Zn(II) and IB proportion is decreased.

Figure 8

Location of His 13 and His 14 in the fibrillar structure of Aβ(1–40). (PDB ID: 2LMN)