Mutants of Metal Binding Site M1 in APP E2 Show Metal Specific Differences in Binding of Heparin but Not of sorLA

Abstract

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease (AD) and appear to be essential for neuronal development and cell homeostasis. Proteolytic processing of APP and its physiological function depend on its interaction with heparin and are influenced by the binding of metal ions and sorLA. We created various mutations of metal binding site M1 residing within the extracellular E2 domain of APP. Using isothermal titration calorimetry and circular dichroism spectroscopy, we analyzed the binding of Cu(2+) and Zn(2+) to APP E2 and identified two mutations that are most suited for functional studies to dissect ion specific effects of metal binding. The H313A mutation abrogates only copper-based effects, whereas the H382A mutation weakens any metal binding at M1 of APP E2. Subsequently, we tested the effect of Cu(2+) and Zn(2+) on the binding of heparin and sorLA to APP E2 using a chromatographic technique and surface plasmon resonance. We show that Zn(2+) and to a larger degree also Cu(2+) enhance the binding of heparin to APP E2, consistent with an extracellular regulation of the function of APP by both metal ions. In contrast, neither ion seemed to affect the interaction between APP E2 and sorLA. This supports an intracellular interaction between the latter two partners that would not sense extracellular variations of metal ions upon synaptic activity.

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein – a lesson in quantification of metal binding to proteinsvialigand competition

“…[32][33][34][35] Metal binding to the M1 site enhances the binding affinity of APP E2 for heparin. 36 These previous observations may help to understand the specificity of APP E2 in forming stable Cu(I)-mediated ternary complexes with the Cu(I) probe ligands that can be disrupted by heparin. Each ligand (Fig.…”

“…25 A further mutation study demonstrated that stable binding of Cu(II) by the M1 site requires coordination by all four His ligands, but only a sub-set of 2-3 for Zn(II). 36 As heparin itself shows no detectable affinity for Cu(I), it is unlikely that the glycosaminoglycan is directly involved in the Cu(I) binding.…”

“…[32][33][34][35] A recent study showed that Cu(II) binding to the M1 site in APP E2 enhances the heparin binding affinity, as does Zn(II) to a lesser extent. 36 Given the abundance of GAGs in the extracellular matrix, it is likely that a high proportion of cell-surface APP is heparinbound. 37 Consequently, the metal-binding properties of the E2/heparin complex are of interest.…”

Copper and zinc ions govern the trans‐directed dimerization of APP family members in multiple ways

“…Interestingly, copper and zinc binding to APP/APLPs has been shown to alter their cis-and trans-directed dimerization properties. Zinc binding to the E2 domain increases the affinity of APP to heparin (Multhaup et al 1995;Dienemann et al 2015) and favors oligomerization of E2 domains from APP and APLP1 but not from APLP2 (Mayer et al 2014). Nevertheless, zinc-mediated oligomerization in the cellular context has been observed for all APP family members and is most pronounced for APLP1 (Mayer et al 2014(Mayer et al , 2016.…”

“…; Dienemann et al . ) and favors oligomerization of E2 domains from APP and APLP1 but not from APLP2 (Mayer et al . ).…”