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, KD'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.