UC Santa Cruz

The cellular prion protein (PrPC) is comprised of two domains – a globular C-terminal domain and an unstructured N-terminal domain. Copper drives 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 glycans of the prion protein are located in close proximity to the C-terminal histidines that we have shown are necessary for the neuroprotective copper tether. The recombinant protein used for the previously described experiments lacked glycans, and we wondered if they would have any effect on the observed interaction. Since the glycans are fairly large and located on the surface of the protein, we thought that they may sterically hinder the cis interaction, falsifying or requiring modification of the interpretation of the previously described results. We set out to create glycosylated protein in order to directly test the impact of these moieties on the protective cis interaction. Bacterially expressed proteins used in NMR studies lack glycans, and proteins from other organisms are neither 15N labelled, nor glycosylated homogenously. We developed a method to add two artificial glycans to uniformly 15N labelled prion protein, using a buffer system that evolves over a pH range in order 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. Using our glycosylated, 15N labelled protein, we tested the effects of the glycans on the protein’s previously observed cis interaction. In opposition to our hypothesis that the glycans would prevent the interaction through steric hindrance, we observed that the interaction was refined by the glycans; the glycans narrow down the region of the C-terminus with which copper interacts. We then found that the presence of glycans partially restores the lost cis interaction observed when C-terminal histidines are mutated away. Together, the work in this dissertation shows that both N and C-terminal histidines of the prion protein bind copper ions, driving a neuroprotective self-regulatory interaction, and that the glycans of the protein refine and strengthen this interaction.