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.

The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A Ê resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A Ê long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO±AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals signi®cant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.