Structural Studies of the Alzheimer’s Amyloid Precursor Protein Copper-binding Domain Reveal How it Binds Copper Ions

doi:10.1016/j.jmb.2006.12.041

Journal of Molecular Biology

Volume 367, Issue 1, 16 March 2007, Pages 148-161

https://doi.org/10.1016/j.jmb.2006.12.041 Get rights and content

Abstract

Alzheimer's disease (AD) is the major cause of dementia. Amyloid β peptide (Aβ), generated by proteolytic cleavage of the amyloid precursor protein (APP), is central to AD pathogenesis. APP can function as a metalloprotein and modulate copper (Cu) transport, presumably via its extracellular Cu-binding domain (CuBD). Cu binding to the CuBD reduces Aβ levels, suggesting that a Cu mimetic may have therapeutic potential. We describe here the atomic structures of apo CuBD from three crystal forms and found they have identical Cu-binding sites despite the different crystal lattices. The structure of Cu²⁺-bound CuBD reveals that the metal ligands are His147, His151, Tyr168 and two water molecules, which are arranged in a square pyramidal geometry. The site resembles a Type 2 non-blue Cu center and is supported by electron paramagnetic resonance and extended X-ray absorption fine structure studies. A previous study suggested that Met170 might be a ligand but we suggest that this residue plays a critical role as an electron donor in CuBDs ability to reduce Cu ions. The structure of Cu⁺-bound CuBD is almost identical to the Cu²⁺-bound structure except for the loss of one of the water ligands. The geometry of the site is unfavorable for Cu⁺, thus providing a mechanism by which CuBD could readily transfer Cu ions to other proteins.

Introduction

Alzheimer's disease (AD) is the leading cause of dementia among the elderly and is characterized by the presence of amyloid plaques, extensive neuronal death and shrinkage of the brain. It is increasingly accepted that the neurotoxic Aβ peptide is responsible for compromising neuronal functions and triggering cell death.¹ The peptide is derived from the cleavage of the amyloid precursor protein (APP)² and is the main constituent of the amyloid plaques. APP belongs to a wider family of APP-like proteins (APLPs) that include APLP1 and APLP2.³ These proteins exhibit functional redundancy to some degree and can undergo cleavage, but only APP cleavage gives rise to the Aβ peptide. The cleavage occurs during trafficking and maturation through the protein secretory pathway by membrane-bound proteinases. Aβ arises through the sequential cleavage by the β-site APP cleaving enzyme (BACE)4., 5. at a site just outside the plasma membrane and the γ-secretase protein complex⁶ within the membrane (Figure 1).

APP is a Type-I transmembrane protein with a large extracellular portion, which can be structurally and functionally subdivided into several domains (Figure 1). At the N terminus is a cysteine-rich region consisting of the growth factor domain (GFD), which binds heparin and can stimulate neurite outgrowth,7., [8] and a copper-binding domain (CuBD) that binds Cu and zinc.⁹ The cysteine-rich region is followed by an acidic-rich region of sequence, a KPI (Kunitz-type protease inhibitor) domain and an OX2 domain.10., 11. The KPI and OX2 domains may be spliced out, to give rise to three (main) variants APP₇₇₀ (with 770 amino acid residues), APP₇₅₁ and APP₆₉₅. APP₆₉₅ is the most common isoform in the brain.¹² Following these domains is a glycosylated domain referred to as E2 in the published structure by Wang and Ha.¹³ Next is a region predicted to adopt no regular secondary structure (unpublished results) and the transmembrane region. The C-terminal cytoplasmic tail may be involved in various cellular functions, such as transcription signaling,¹⁴ through interaction with a multitude of proteins.¹⁵ However, an endogenous ligand that triggers signaling through APP has yet to be identified. There is evidence that APP exists in a monomer to dimer equilibrium in the membrane.[16], 17. The binding of high molecular weight heparin also appears able to induce dimerization.¹⁸ Dimerization may cause an initiation of signaling events¹⁹ and play a role in modulating APP cleavage as the production of Aβ, in particular dimeric Aβ, is increased when APP is forcibly dimerized.¹⁶ What influences the monomer-dimer equilibrium is not clear, but certain pathological states such as heme deficiency²⁰ or even Aβ itself²¹ may lead to greater APP oligomerization.

APP is a metalloprotein that contributes to Cu ion metabolism. APP binds to free Cu²⁺ ions and once bound, Cu²⁺ is reduced to Cu⁺ in vitro.²² This can lead to increased Cu-mediated neurotoxicity in cultured neurons,²³ presumably through increased oxidative stress or the generation of reactive oxygen species.²⁴ A role for APP in Cu efflux is supported by a series of experiments that demonstrate an increased intracellular accumulation of Cu in the brains of APP/APLP2 double knockout mice²⁵ and neuronal cultures²⁶ whilst transgenic over-expression of APP has the opposite effects in mice and cultured neurons.[26], 27. The involvement of CuBD in Cu efflux has been demonstrated in yeast expressing an APP ectodomain fragment. Mutating the CuBD significantly increases intracellular Cu levels compared to yeast expressing a wild-type fragment.²⁸

The interaction between Cu ions and APP, via CuBD, can modulate Aβ production and the progression of AD. The treatment of CHO cells over-expressing APP with extracellular Cu²⁺ leads to reduced Aβ production and a shift of the cleavage equilibrium away from the BACE/γ-secretase pathway.²⁹ The effects are abolished when the Cu binding residues within CuBD are mutated.³⁰ The beneficial outcomes of Cu²⁺ binding to APP have been illustrated in two independent transgenic mouse studies. By supplementing drinking water with Cu²⁺ ions, the survival of APP23 mice (which overproduce Aβ) was improved along with increased bioavailable Cu²⁺ and reduced Aβ levels in the brain.³¹ Similar effects were achieved by crossing TgCRND8 mice (that overproduce Aβ) with mice that have raised brain Cu levels due to the expression of a mutant Cu transporter CuATPase7b. These mice had decreased amyloid plaque and plasma Aβ levels.³²

To define the molecular interaction between the APP CuBD and Cu, we have determined the crystal structures of CuBD in apo and Cu-bound forms at near atomic resolution. We have also performed electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) experiments to probe the Cu binding site in solution. The structural studies reveal how Cu binds to APP and provide the basis for the design of novel therapeutics to combat AD.

Section snippets

Crystal structures of Apo CuBD

Although we had previously determined a structure of CuBD in apo form by NMR,⁹ we wished to obtain a higher resolution structure of the Cu binding site in the apo protein that we could compare to Cu-bound crystal structures of CuBD. We have obtained three different crystal forms, named Type A, B and C, consisting of 11 different views of the molecule, all diffracting to high resolution. Hence any crystal lattice effects on the Cu binding site could readily be discerned. All the Cu binding

Discussion

We have determined the structure of apo CuBD in three different crystal forms at high resolution with 11 different views of the molecule. The structure consists of an α-helix packed against a three-strand β-sheet. According to the classification by the CATH server,³⁸ CuBD belongs to the class of mixed α/β proteins and displays an architecture of a two-layered sandwich. The topology of CuBD is classified as either defensin A-like proteins or α-β plaits. Proteins that exhibit the latter topology

Apo CuBD_133-189 (Type A) structure

This construct consisted of residues 133 to 189 plus a glutamine and an alanine at the N terminus as a result of a cloning artifact. The expression, purification and crystallization of this construct have been described in detail elsewhere.⁵⁵ In brief, the protein was expressed in Pichia pastoris, purified by gel filtration chromatography and concentrated to ∼10 mg ml⁻¹. The protein was crystallized by the hanging drop method at room temperature in 0.1 M Hepes (pH 8.0), 28%–32% (w/v) PEG 10 000.

Acknowledgements

We thank Galina Polekhina for general advice on crystallography, and Harry Tong and other BioCARS staff for their help with data collection at APS. Use of APS and SSRL was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical

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¹: Present address: G. K.-W. Kong, Abt. Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, Am Klopferspitz 18, D-82152 Martinsried, Germany; J. J. Adams, Australian Synchrotron Project, 800 Blackburn Road, Clayton, Victoria 3168, Australia; W. J. McKinstry, Pfizer Animal Health, Parkville, Victoria 3052, Australia.

^†: Co-senior authors.

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Journal of Molecular Biology

Structural Studies of the Alzheimer’s Amyloid Precursor Protein Copper-binding Domain Reveal How it Binds Copper Ions

Abstract

Introduction

Section snippets

Crystal structures of Apo CuBD

Discussion

Apo CuBD133-189 (Type A) structure

Acknowledgements

Neurochem. Intl.

Trends Cell Biol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Mol. Cell

J. Biol. Chem.

J. Mol. Biol.

FEBS Letters

Biochem. Pharmacol.

Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

In Methods Enzymol.

J. Inorg. Biochem.

Alzheimer's disease is a synaptic failure

Science

The presursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor

Nature

Purification and cloning of amyloid precursor protein β-secretase from human brain

Nature

Membrane-anchored aspartyl protease with Alzheimer's disease β-secretase activity

Nature

Crystal structure of the N-terminal, growth factor-like domain of Alzheimer amyloid precursor protein

Nature Struct. Biol.

Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease

Nature

Processing of beta-amyloid precursor protein in microglia and astrocytes favors an internal localization over constitutive secretion

J. Neurosci.

A transcriptively active complex of APP with Fe65 and histone acetyltransferase Tip60

Science

Proteolytic processing and cell biological functions of the amyloid precursor protein

J. Cell Sci.

Homo- and heterodimerization of APP family members promotes intercellular adhesion

EMBO J.

Amyloid precursor protein mediates proinflammatory activation of monocytic lineage cells

J. Biol. Chem.

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging

Proc. Natl Acad. Sci. USA

Amyloid β protein toxicity mediated by the formation of amyloid-β protein precursor complexes

Annals Neurol.

Apo CuBD_133-189 (Type A) structure