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.

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. 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 divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.

Little is known about the effect of conformation on passive membrane diffusion rates in small molecules. Evidence suggests that intramolecular hydrogen bonding may play a role by reducing the energetic cost of desolvating hydrogen bond donors, especially amide N-H groups. We set out to test this hypothesis by investigating the passive membrane diffusion characteristics of a series of cyclic peptide diastereomers based on the sequence cyclo[Leu-Leu-Leu-Leu-Pro-Tyr]. We identified two cyclic hexapeptide diastereomers based on this sequence, whose membrane diffusion rates differed by nearly two log units. Results of solution NMR studies and hydrogen/deuterium (H/D) exchange experiments showed that membrane diffusion rates correlated with the degree of intramolecular hydrogen bonding and H/D exchange rates. The most permeable diastereomer, cyclo[d-Leu-d-Leu-Leu-d-Leu-Pro-Tyr] (1), exhibited a passive membrane diffusion rate comparable to that of the orally available drug cyclosporine A.

The prion protein (PrP) binds divalent copper at physiologically relevant conditions and is believed to participate in copper regulation or act as a copper-dependent enzyme. Ongoing studies aim at determining the molecular features of the copper binding sites. The emerging consensus is that most copper binds in the octarepeat domain, which is composed of four or more copies of the fundamental sequence PHGGGWGQ. Previous work from our laboratory using PrP-derived peptides, in conjunction with EPR and X-ray crystallography, demonstrated that the HGGGW segment Copper coordination arises from the His imidazole and sequential deprotonated glycine amides. In this present work, recombinant, full-length Syrian hamster PrP is investigated using EPR methodologies. Four copper ions are taken up in the octarepeat domain, which supports previous findings. However, quantification studies reveal a fifth binding site in the flexible region between the octarepeats and the PrP globular C-terminal domain. A series of PrP peptide constructs show that this site involves His96 in the PrP(92-96) segment GGGTH. Further examination by X-band EPR, S-band EPR, and electron spin-echo envelope spectroscopy, demonstrates coordination by the His96 imidazole and the glycine preceding the threonine. The copper affinity for this type of binding site is highly pH dependent, and EPR studies here show that recombinant PrP loses its affinity for copper below pH 6.0. These studies seem to provide a complete profile of the copper binding sites in PrP and support the hypothesis that PrP function is related to its ability to bind copper in a pH-dependent fashion.Prion diseases are fatal neurodegenerative disorders of both humans and animals (1). The causative agent is an isoform of a normal, host-encoded membrane glycoprotein called the prion protein (PrP). 1 The normal cellular isoform (PrP C ) is the precursor to the pathogenic, protease-resistant isoform termed PrP Sc , which is responsible for homologous pathologies within its individual hosts. With rare but notable exceptions, prion diseases respect the species barrier (2,3). Among these exceptions is the transmission of disease from scrapie-infected † This work was supported by NIH Grants GM 65790 (G.L.M.), GM 60609 (G.J.G.), GM 40168 (J.P.), AG02132 and AG10770 (S.B.P.).

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as β-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors. AUTHORS' SUMMARYThe marked spectrum of color and diversity of patterns that we see in mammals arises, unexpectedly, from variation in the quantity, quality, and regional distribution of just two types of pigment-black eumelanin and yellow pheomelanin. The appeal of unusual coat colors and patterns has motivated their selection in domestic animals, providing geneticists with a model for studying gene action and interaction that began a century ago and continues today. Most of the work has been carried out in laboratory mice, where studies of more than 100 different coat-color mutations have provided insight into stem cell biology (hair graying), biogenesis of intracellular organelles (pigmentary dilution), and hormonereceptor interactions (switching between the synthesis of eumelanin and pheomelanin). ‡ To whom correspondence should be addressed. gbarsh@stanford.ed. * These authors contributed equally to the work. The latter process-commonly known as pigment "type-switching"-is controlled primarily by the melanocortin system, in which a family of G protein-coupled receptors (identified by virtue of their response to α-melanocyte-stimulating hormone or adrenocorticotrophic hormone) has been implicated not only in pigmentation but also in cortisol production, body weight regulation, and exocrine gland secretion. In most mammals, pigment type-switching is controlled by two genes, the Melanocortin 1 receptor (Mc1r) and Agouti, which encode a seven transmembrane-domain receptor and its extracellular ligand, respectively. Indeed, our current understanding of melanocortin biology stems from the identification in laboratory mice of Mc1r mutations as the cause of recessive yellow and Agouti mutations as the cause of lethal yellow.Clarence Cook Little, who developed many of the original laboratory mouse strains and founded The Jackson Laboratory, was also one of the first dog geneticists. He recognized that dominant inheritance of a black coat was mediated differently in dogs than in other animals (1). Using classical linkage analysis, we realized that the dominant black gene represented a previously unrecognized component of the melanocortin pathway (2). Unexpectedly, we found ...

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Alpha-synuclein (SNCA) is central to the pathogenesis of Parkinson disease (PD), with 3 missense mutations reported to date. We report a novel mutation (p.H50Q) in a pathologically proven case.

A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt-Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for Cu(2+). The structural features of the Cu(2+) binding sites have now been characterized and are providing important clues about the normal function of the prion protein and perhaps how metals or loss of protein function play a role in disease. The link between prion protein and copper may provide insight into the general, and recently appreciated, role of metals in neurodegenerative disease.