Molecular Features of the Zn²⁺ Binding Site in the Prion Protein Probed by ¹¹³Cd NMR

The cellular prion protein (PrP^C) is a zinc-binding protein that contributes to the regulation of Zn²⁺ and other divalent species of the central nervous system. Zn²⁺ coordinates to the flexible, N-terminal repeat region of PrP^C and drives a tertiary contact between this repeat region and a well-defined cleft of the C-terminal domain. The tertiary structure promoted by Zn²⁺ is thought to regulate inherent PrP^C toxicity. Despite the emerging consensus regarding the interaction between Zn²⁺ and PrP^C, there is little direct spectroscopic confirmation of the metal ion's coordination details. Here, we address this conceptual gap by using Cd²⁺ as a surrogate for Zn²⁺. NMR finds that Cd²⁺ binds exclusively to the His imidazole side chains of the repeat segment, with a dissociation constant of ∼1.2 mM, and promotes an N-terminal-C-terminal cis interaction very similar to that observed with Zn²⁺. Analysis of ¹¹³Cd NMR spectra of PrP^C, along with relevant control proteins and peptides, suggests that coordination of Cd²⁺ in the full-length protein is consistent with a three- or four-His geometry. Examination of the mutation E199K in mouse PrP^C (E200K in humans), responsible for inherited Creutzfeldt-Jakob disease, finds that the mutation lowers metal ion affinity and weakens the cis interaction. These findings not only provide deeper insight into PrP^C metal ion coordination but they also suggest new perspectives on the role of familial mutations in prion disease.