title: Molecular features of the copper binding sites in the octarepeat domain of the prion protein.
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 15N-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 approximately 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
authors.forename: Colin S
authors.initials: CS
authors.lastname: Burns
authors.forename: Eliah
authors.initials: E
authors.lastname: Aronoff-Spencer
authors.forename: Christine M
authors.initials: CM
authors.lastname: Dunham
authors.forename: Paula
authors.initials: P
authors.lastname: Lario
authors.forename: Nikolai I
authors.initials: NI
authors.lastname: Avdievich
authors.forename: William E
authors.initials: WE
authors.lastname: Antholine
authors.forename: Marilyn M
authors.initials: MM
authors.lastname: Olmstead
authors.forename: Alice
authors.initials: A
authors.lastname: Vrielink
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Gerfen
authors.forename: Jack
authors.initials: J
authors.lastname: Peisach
authors.forename: William G
authors.initials: WG
authors.lastname: Scott
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Millhauser
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D020449:Repetitive Sequences, Amino Acid
chemical_list: D011328:Prions; D003300:Copper