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The cellular prion protein (PrPC) comprises two domains: a globular C-terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary contact in PrPC, inducing a neuroprotective cis interaction that structurally links the protein's two domains. The location of this interaction on the C terminus overlaps with the sites of human pathogenic mutations and toxic antibody docking. Combined with recent evidence that the N terminus is a toxic effector regulated by the C terminus, there is an emerging consensus that this cis interaction serves a protective role, and that the disruption of this interaction by misfolded PrP oligomers may be a cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein's cis interaction, which helps to protect against neurotoxicity carried out by its N terminus. We show that simultaneous mutation of these histidines drastically weakens the cis interaction and enhances spontaneous cationic currents in cultured cells, the first C-terminal mutant to do so. Whereas previous studies suggested that Cu2+ coordination was localized solely to the protein's N-terminal domain, we find that both domains contribute equatorially coordinated histidine residue side-chains, resulting in a novel bridging interaction. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by sequestering copper from the C terminus. Our findings further establish a structural basis for PrPC's C-terminal regulation of its otherwise toxic N terminus.

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.

Copper plays a critical role in prion protein (PrP) physiology. Cu(2+) binds with high affinity to the PrP N-terminal octarepeat (OR) domain, and intracellular copper promotes PrP expression. The molecular details of copper coordination within the OR are now well characterized. Here we examine how Cu(2+) influences the interaction between the PrP N-terminal domain and the C-terminal globular domain. Using nuclear magnetic resonance and copper-nitroxide pulsed double electron-electron resonance, with molecular dynamics refinement, we localize the position of Cu(2+) in its high-affinity OR-bound state. Our results reveal an interdomain cis interaction that is stabilized by a conserved, negatively charged pocket of the globular domain. Interestingly, this interaction surface overlaps an epitope recognized by the POM1 antibody, the binding of which drives rapid cerebellar degeneration mediated by the PrP N terminus. The resulting structure suggests that the globular domain regulates the N-terminal domain by binding the Cu(2+)-occupied OR within a complementary pocket.

The structures and relative free energies in aqueous solution of the Cu(II) complexes of the 'histidine walk' peptides, [AcHGGGNH.sub.2], [AcGHGGNH.sub.2], [AcGGHGNH.sub.2], and [AcGGGHNH.sub.2], were determined as a function of pH. Numerous structures of each species were found by gaseous- and solution-phase geometry optimization at the B3LYP/6-31G(d) level, and the effect of solvation estimated by the IEFPCM continuum solvation model. Free energies of solvation of the ionic species are large and favour structures with an extended peptide chain. In all Cu(II)-peptide complexes, deprotonation of two amide groups occurs readily at or below pH 7. In each system, the most abundant species at pH 7 is a neutral 1:1 complex with N3O1 coordination pattern. Binding in the forward direction toward the C terminus is preferred. The results are compared to recent experimental spectroscopic and potentiometric studies on related systems. Alternative explanations are offered for some of the experimental observations. Key words: copper-peptides, theoretical, DFT, prion, binding affinities, CD spectra. On a determine les structures et les energies libres, en solutions aqueuses et en fonction du pH, des complexes de Cu(II) des peptides de la serie histidine, [AcHGGGNH.sub.2], [ACGHGGNH.sub.2], [AcGGHGNH.sub.2] et [ACGGGHNH.sub.2]. On a determine les diverses structures de chacune des especes, en phase gazeuse et en solution, par une optimisation de la geometrie au niveau B3LYP/6-31G(d) et on a evalue l'effet de solvatation par le modele de solvatation continue IEEPCM. Les energies libres de solvatation des especes ioniques sont importantes et elles favorisent les structures avec une chaine peptidique etendue. Dans tous les complexes Cu(II)-peptide, la deprotonation de deux groupes amides se produit facilement a des pH egal ou inferieurs a 7. Dans chaque systeme, l'espece la plus abondante au pH 7 est un complexe 1:1 neutre comportant un patron de coordination N3O1. La fixation est favorisee dans la direction allant vers la position C-terminale. On compare les resultats obtenus ici avec ceux rapportes recemment pour des etudes experimentales spectroscopiques et potentiome triques sur des systemes apparentes. Des explications alternatives sont proposees pour un certain nombre d'observations experimentales. Mots-cles : complexes cuivre-peptides, theorique, theorie de la fonctionnelle de densite (TFD), affinites de fixation, spectres de dichroisme circulaire (DC). [Traduit par la Redaction], Introduction The pathology of many neurodegenerative diseases involves the disruption of normal trafficking of a metal ion which is ultimately related to its binding to a biologically prevalent protein or [...]

The N-terminal domain of mammalian prion proteins contains several tandem repeats of the octapeptide PHGGGWGQ, each one capable of selectively binding up to 1 equiv of [Cu.sup.2+]. Under saturating conditions [Cu.sup.2+] is known to coordinate the HGG portion of the repeat sequence via the histidine imidazole side chain, two deprotonated amide N-atoms, and a backbone carbonyl O-atom. Using appropriate selection criteria, we have generated a short list of candidate metal ions ([Co.sup.3+], [Ni.sup.2+], [Pd.sup.2+], [Pt.sup.2+]) that can serve as potential surrogates for [Cu.sup.2+]. The selected metal ions were screened for binding interactions with the OR-derived peptide fragment [AcHGGGWNH.sub.2] (Ac = acetyl, amino acid residues in italics) using electrospray ionization mass spectrometry. The coordination geometries of these metal ions with the synthetic OR peptide were subsequently determined from fragment analysis using collision-induced dissociation tandem mass spectrometry. Our results indicate that, although [Co.sup.3+], [Pd.sup.2+], and [pt.sup.2+] all bind to the OR fragment via the peptide backbone to varying extents, each of these metal ions appears to associate with the peptide in a unique manner, which is distinct from the way in which [Cu.sup.2+] is coordinated. This work illustrates the extremely strong selectivity for [Cu.sup.2+] of this highly conserved region of the mammalian prion protein.

Objective: Multiple sclerosis (MS) is a heterogeneous inflammatory demyelinating disease. Iron distribution is altered in MS patients' brains, suggesting iron liberation within active lesions amplifies demyelination and neurodegeneration. Whether the amount and distribution of iron are similar or different among different MS immunopatterns is currently unknown. Methods: We used synchrotron X‐ray fluorescence imaging, histology, and immunohistochemistry to compare the iron quantity and distribution between immunopattern II and III early active MS lesions. We analyzed archival autopsy and biopsy tissue from 21 MS patients. Results: Immunopattern II early active lesions contain 64% more iron (95% confidence interval [CI] = 17–127%, p = 0.004) than immunopattern III lesions, and 30% more iron than the surrounding periplaque white matter (95% CI = 3–64%, p = 0.03). Iron in immunopattern III lesions is 28% lower than in the periplaque white matter (95% CI = −40 to −14%, p < 0.001). When normalizing the iron content of early active lesions to that of surrounding periplaque white matter, the ratio is significantly higher in immunopattern II (p < 0.001). Microfocused X‐ray fluorescence imaging shows that iron in immunopattern II lesions localizes to macrophages, whereas macrophages in immunopattern III lesions contain little iron. Interpretation: Iron distribution and content are heterogeneous in early active MS lesions. Iron accumulates in macrophages in immunopattern II, but not immunopattern III lesions. This heterogeneity in the two most common MS immunopatterns may be explained by different macrophage polarization, origin, or different demyelination mechanisms, and paves the way for developing new or using existing iron‐sensitive magnetic resonance imaging techniques to differentiate among immunopatterns in the general nonbiopsied MS patient population. ANN NEUROL 2021;89:498–510 [ABSTRACT FROM AUTHOR]

Serine and alanine variants at position 185 of recombinant chicken SO were generated to better understand the role of reside Cys in the catalytic mechanism of sulfite oxidase. The crystal structures of C185S and C185A variants which contain a fully oxidized, trioxo form of the cofactor, and Tyr322 could undergo a conformational change that is relevant to the reaction mechanism.

Diffusible ions (Na+, K+, Mg2+, Ca2+, Cl–) are vital for healthy function of all cells, especially brain cells. Unfortunately, the diffusible nature of these ions renders them difficult to study with traditional microscopy in situ within ex vivo brain tissue sections. This mini-review examines the recent progress in the field, using direct elemental mapping techniques to study ion homeostasis during normal brain physiology and pathophysiology, through measurement of ion distribution and concentration in ex vivo brain tissue sections. The mini-review examines the advantages and limitations of specific techniques: proton induced X-ray emission (PIXE), X-ray fluorescence microscopy (XFM), secondary ion mass spectrometry (SIMS), laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and the sample preparation requirements to study diffusible ions with these methods. [ABSTRACT FROM AUTHOR]