Copper binding alters the core structure of amyloid fibrils formed by Y145Stop human prion protein

Abstract

Transmissible spongiform encephalopathies (or prion diseases) such as Creutzfeldt-Jacob disease, mad cow disease, and scrapie are characterized by accumulation in the brain of misfolded prion protein aggregates (PrP^Sc) that have properties of amyloid fibrils. Given that transition metal ions, such as copper and zinc, appear to be important for physiological functions of cellular PrP (PrP^C) as well as for prion disease pathogenesis, exploring their role in the protein aggregation process is of considerable interest. Copper(II) in particular is well-known to bind to the four tandem octapeptide repeats (PHGGGWGQ) located in the N-terminal region of PrP (human PrP amino acids 60–91), as well as to additional histidine binding sites outside the octarepeat region with distinct binding modes depending on Cu²⁺ concentration. Here, using the Y145Stop human prion protein variant (huPrP23-144) as a model and a combination of multidimensional solution and solid-state NMR spectroscopy, atomic force microscopy and thioflavin T fluorescence assays we probed the binding of Cu²⁺ to monomeric huPrP23-144 and the impact of this binding on fibril assembly kinetics and their structural properties. Remarkably, we found that fibrils formed by huPrP23-144 containing one molar equivalent of bound Cu²⁺ adopt a core structure that is distinct from that found for huPrP23-144 in the absence of Cu²⁺ but, instead, corresponds to a conformational strain formed by huPrP23-144 containing the A117V mutation. A similar huPrP23-144 A117V-like amyloid core structure was adopted by a Cu²⁺-bound Δ51-91 huPrP23-144 deletion variant lacking the entire octarepeat region, suggesting that Cu²⁺ binding to His residues 96, 111 and 140 located near the C-terminus of huPrP23-144 is primarily responsible for the observed change in fibril conformation, potentially due to partial structuring of the intrinsically disordered huPrP23-144 by the bound Cu²⁺ during the fibril assembly process. We also found that fibrils formed by Cu²⁺-bound huPrP23-144 adopt the native huPrP23-144-like rather than A117V-like structure when the fibrillization reaction is seeded with pre-formed huPrP23-144 amyloid.