High Affinity Binding between Copper and Full-length Prion Protein Identified by Two Different Techniques

Andrew R Thompsett,Salama R Abdelraheim,Maki Daniels,David R Brown

doi:10.1074/jbc.m506521200

Andrew R Thompsett, Salama R Abdelraheim + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m506521200

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Abstract

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.

Highlights

As a result, knowledge of copper binding to PrP is important in considering both the normal function of PrPc and its influence in prion diseases
The thermodynamics of Cu(II) binding has been examined by isothermal titration calorimetry (ITC)2 to measure affinity
Analysis Using Isothermal Titration Calorimetry—The thermodynamic properties of ligand-macromolecule interactions can be accurately determined by ITC

Summary

Introduction

Knowledge of copper binding to PrP is important in considering both the normal function of PrPc and its influence in prion diseases. Study of a peptide with amino acid residues 23–98 of PrPc suggested that this fragment could bind 5 Cu(II) atoms [9] Four of these sites were designated as belonging to the octameric repeat region, the location of the so-called fifth site has been difficult to identify. It has been suggested that the alternative Cu(II) binding site has higher affinity for Cu(II) than the octameric repeat region [11, 15] None of these studies has estimated the relative affinity of these sites within full-length PrP and has relied on peptides and fragments. Where possible full-length protein PrP has been utilized to avoid the discrepancies associated with peptides, and Cu(II) has been added in a chelated form to mimic physiological conditions

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