Molecular features of the copper binding sites in the octarepeat domain of the prion protein

Colin S. Burns, Eliah Aronoff-Spencer, Christine M. Dunham, Paula Lario, Nikolai I. Avdievich, William E. Antholine, Marilyn M. Olmstead, Alice Vrielink, Gary J. Gerfen, Jack Peisach, William G. Scott, Glenn L. Millhauser

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Abstract

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an l5N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH ≈6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.

Original languageEnglish (US)
Pages (from-to)3991-4001
Number of pages11
JournalBiochemistry
Volume41
Issue number12
DOIs
StatePublished - Mar 26 2002

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Copper
Binding Sites
Paramagnetic resonance
Crystal structure
Molecular recognition
Peptides
Biochemistry
Prions
Prion Proteins
Conserved Sequence
Endosomes
Endocytosis
Histidine
Glycine
Libraries
Extracellular Matrix
Spectrum Analysis
Spectroscopy
Water
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Burns, C. S., Aronoff-Spencer, E., Dunham, C. M., Lario, P., Avdievich, N. I., Antholine, W. E., ... Millhauser, G. L. (2002). Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry, 41(12), 3991-4001. https://doi.org/10.1021/bi011922x

Molecular features of the copper binding sites in the octarepeat domain of the prion protein. / Burns, Colin S.; Aronoff-Spencer, Eliah; Dunham, Christine M.; Lario, Paula; Avdievich, Nikolai I.; Antholine, William E.; Olmstead, Marilyn M.; Vrielink, Alice; Gerfen, Gary J.; Peisach, Jack; Scott, William G.; Millhauser, Glenn L.

In: Biochemistry, Vol. 41, No. 12, 26.03.2002, p. 3991-4001.

Research output: Contribution to journalArticle

Burns, CS, Aronoff-Spencer, E, Dunham, CM, Lario, P, Avdievich, NI, Antholine, WE, Olmstead, MM, Vrielink, A, Gerfen, GJ, Peisach, J, Scott, WG & Millhauser, GL 2002, 'Molecular features of the copper binding sites in the octarepeat domain of the prion protein', Biochemistry, vol. 41, no. 12, pp. 3991-4001. https://doi.org/10.1021/bi011922x
Burns CS, Aronoff-Spencer E, Dunham CM, Lario P, Avdievich NI, Antholine WE et al. Molecular features of the copper binding sites in the octarepeat domain of the prion protein. Biochemistry. 2002 Mar 26;41(12):3991-4001. https://doi.org/10.1021/bi011922x
Burns, Colin S. ; Aronoff-Spencer, Eliah ; Dunham, Christine M. ; Lario, Paula ; Avdievich, Nikolai I. ; Antholine, William E. ; Olmstead, Marilyn M. ; Vrielink, Alice ; Gerfen, Gary J. ; Peisach, Jack ; Scott, William G. ; Millhauser, Glenn L. / Molecular features of the copper binding sites in the octarepeat domain of the prion protein. In: Biochemistry. 2002 ; Vol. 41, No. 12. pp. 3991-4001.
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abstract = "Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an l5N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH ≈6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.",
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AU - Avdievich, Nikolai I.

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AU - Vrielink, Alice

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AU - Millhauser, Glenn L.

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N2 - Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an l5N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH ≈6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.

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