Location and properties of metal-binding sites on the human prion protein

Jackson, Graham S.; Murray, Ian V.J.; Hosszu, Laszlo L. P.; Gibbs, Nicholas; Waltho, Jonathan P.; Clarke, Anthony R.; Collinge, John

doi:10.1073/pnas.151038498

Cited by 463 publications

(553 citation statements)

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“…As shown in our previous work, EPR spectra from a copper titration of PrP (23)(24)(25)(26)(27)(28) show a progression of different spectra and these are assigned to individual binding components (17). Component 3 dominates at low copper occupancy (≤1.0 equiv).…”

Section: Cooperativity Of Cu 2+ Bindingsupporting

confidence: 57%

“…Clearly, the results reported here require not only confirmation in full-length PrP but also an assessment of the influence of non-octarepeat coordination sites (20,28) and other features that may affect Cu 2+ uptake. With regard to octarepeat coordination, however, our previously published experiments on full-length PrP Since the discovery of the physiological connection of PrP to copper, there have been intensive efforts to determine the precise Cu 2+ affinity (8,10,(21)(22)(23)(24)(25). As noted in the Introduction, there is wide disagreement with reported K d values distributed over nearly 8 orders of magnitude.…”

Section: Discussionmentioning

confidence: 99%

“…For an enzyme, one would expect high affinity, reflected through a low dissociation constant (K d ) (21), whereas buffering or sensing would require a lower affinity, with a K d near the concentration of extracellular copper (22). Unfortunately, investigations into the dissociation constant and the mechanism of copper uptake reveal highly disparate outcomes depending upon the measurement technique and the particular PrP construct (8,10,(21)(22)(23)(24)(25). Current estimates place K d between 10 −6 M (10,22,23) and 10 −14 M (21,24), an experimental uncertainty of 8 orders of magnitude.…”

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confidence: 99%

“…Unfortunately, investigations into the dissociation constant and the mechanism of copper uptake reveal highly disparate outcomes depending upon the measurement technique and the particular PrP construct (8,10,(21)(22)(23)(24)(25). Current estimates place K d between 10 −6 M (10,22,23) and 10 −14 M (21,24), an experimental uncertainty of 8 orders of magnitude. For example, mass spectrometry studies on PrP(57-91), encompassing all of the octarepeats, finds a dissociation constant of 0.2 μM for the first bound Cu 2+ , with the K d increasing to 12 μM for the fourth Cu 2+ (10).…”

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confidence: 99%

“…Similar micromolar affinities have been identified using amino acid competition studies, as detected by circular dichroism (CD) (23). Alternatively, binding curves determined from tryptophan fluorescence quenching from Cu 2+ report a very high-affinity site with a K d of 8 × 10 −15 M (8 fM) in PrP(57-98) (24). Isothermal titration calorimetry (ITC) and competitive metal capture analysis (CMCA) also support the presence of a high-affinity Cu 2+ -binding site in the octarepeat region (21).…”

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confidence: 99%

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The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

2006

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The prion protein (PrP) binds Cu 2+ in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu 2+ to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu 2+ with a dissociation constant of 0.10 (±0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 μM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.The prion protein (PrP) 1 is responsible for a novel class of infectious, neurodegenerative diseases collectively known as the transmissible spongiform encephalopathies (TSEs) (1-3). The TSEs include scrapie in sheep, mad cow disease (bovine spongiform encephalopathy, BSE), chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. The normal cellular form of the prion protein, referred to as PrP C , is found in a wide range of tissues of all mammalian and avian species. A misfolding event converts the protein to the infectious, β-sheet-rich scrapie form (PrP Sc ) responsible for the TSEs.PrP C is a GPI-anchored glycoprotein expressed in abundance on the surface of neurons, predominantly on presynaptic membranes (4,5). The mature form of PrP, consisting of residues 23-231 in hamster, has a globular C-terminal domain and a largely unstructured N-terminal domain (6) (Figure 1). The C-terminal domain (residues 125-231) contains three α helices, two of which are stabilized by a single interhelical disulfide bond, and a small antiparallel β sheet. The flexible N-terminal domain is glycine-rich and highly flexible. Within the flexible N-terminal region of PrP is the octarepeat domain, composed of four or five highly conserved, contiguous repeats of the eight-residue sequence PHGGGWGQ. The physiological function

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Section: Cooperativity Of Cu 2+ Bindingsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

2006

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Heavy Metal‐binding Proteins and Peptides

2003

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Introduction Historical Outline Chemical Structures Nomenclature and Structure of Metallothioneins Phytochelatins and Phytochelatin–Metal Complexes Structural Properties of Metallochaperones Chemical Analysis and Detection Metallothioneins Phytochelatins Occurrence Metallothioneins Phytochelatins Metallochaperones Functions Metal Homeostasis and the Role of Metallochaperones Buffering and Detoxification Physiology Metallothionein Localization and Isoforms Localization and Compartmentation of Phytochelatin Synthesis Biochemistry Metal‐binding Characteristics of Metallothioneins Biochemistry of Phytochelatin Synthesis Molecular Genetics Metallothionein Genes and Their Regulation Phytochelatin Synthase Genes Biotechnological Applications Patents Outlook and Perspectives

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Prions

Béringue

2015

Reviews in Cell Biology and Molecular Medicine

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Location and properties of metal-binding sites on the human prion protein

Cited by 463 publications

References 23 publications

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

Heavy Metal‐binding Proteins and Peptides

Prions

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