Identification of the Cu2+ Binding Sites in the N-Terminal Domain of the Prion Protein by EPR and CD Spectroscopy

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• 作者：Eliah Aronoff-Spencer ; Colin S. Burns ; Nikolai I. Avdievich ; Gary J. Gerfen ; Jack Peisach ; William E. Antholine ; Haydn L. Ball ; Fred E. Cohen ; Stanley B. Prusiner ; and Glenn L. Millhauser
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：November 14, 2000
• 年：2000
• 卷：39
• 期：45
• 页码：13760 - 13771
• 全文大小：139K
• 年卷期：v.39,no.45(November 14, 2000)
• ISSN：1520-4995

文摘

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism inthe central nervous system. The N-terminal region of human PrP contains four sequential copies of thehighly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectivelybinds divalent copper ions (Cu²⁺) in vivo. To elucidate the specific mode and site of binding, we havestudied a series of Cu²⁺-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequencyS-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observedwhere the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copperion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstratethat the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW givesEPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeatsequences and may therefore comprise the fundamental Cu²⁺ binding unit. Both EPR and CD titrationexperiments demonstrate rigorously a 1:1 Cu²⁺/octarepeat binding stoichiometry regardless of the numberof octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates thatall of the bound Cu²⁺ is detected thereby ruling out strong exchange coupling that is often found whenthere is imidazolate bridging between paramagnetic metal centers. A model consistent with these data isproposed in which Cu²⁺ is bound to the nitrogen of the histidine imidazole side chain and to two nitrogensfrom sequential glycine backbone amides.