N-glycosylation is a potent regulator of prion protein neurotoxicity.

The C-terminal domain of the cellular prion protein (PrPC) contains two N-linked glycosylation sites, the occupancy of which impacts disease pathology. In this study, we demonstrate that glycans at these sites are required to maintain an intramolecular interaction with the N-terminal domain, mediated through a previously identified copper-histidine tether, which suppresses the neurotoxic activity of PrPC. NMR and electron paramagnetic resonance spectroscopy demonstrate that the glycans refine the structure of the protein's interdomain interaction. Using whole-cell patch-clamp electrophysiology, we further show that cultured cells expressing PrP molecules with mutated glycosylation sites display large, spontaneous inward currents, a correlate of PrP-induced neurotoxicity. Our findings establish a structural basis for the role of N-linked glycans in maintaining a nontoxic, physiological fold of PrPC.

Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system.

Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

Production of Artificially Doubly Glycosylated, 15N Labeled Prion Protein for NMR Studies Using a pH-Scanning Volatile Buffer System.

Bacterially expressed proteins used in NMR studies lack glycans, and proteins from other organisms are neither 15N labeled nor glycosylated homogeneously. Here, we add two artificial glycans to uniformly 15N labeled prion protein using a buffer system that evolves over a pH range to accommodate the conflicting pH requirements of the substrate and enzymes without the need to fine-tune buffer conditions. NMR and CD spectroscopy of the protein indicates that the glycans do not influence its fold.

Both N-Terminal and C-Terminal Histidine Residues of the Prion Protein Are Essential for Copper Coordination and Neuroprotective Self-Regulation.

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.