Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Abstract

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu²⁺ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu²⁺ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu²⁺ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu²⁺ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu²⁺ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu²⁺ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu²⁺ ions to the extended peptides at pH 7.4, K_D's are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu²⁺ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu²⁺ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu²⁺, as judged by circular dichroism, Cu²⁺ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu²⁺ transporter by binding Cu²⁺ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.