Zinc drives a tertiary fold in the prion protein with familial disease mutation sites at the interface

Abstract

The cellular prion protein PrP(C) consists of two domains--a flexible N-terminal domain, which participates in copper and zinc regulation, and a largely helical C-terminal domain that converts to β sheet in the course of prion disease. These two domains are thought to be fully independent and noninteracting. Compelling cellular and biophysical studies, however, suggest a higher order structure that is relevant to both PrP(C) function and misfolding in disease. Here, we identify a Zn²⁺-driven N-terminal to C-terminal tertiary interaction in PrP(C). The C-terminal surface participating in this interaction carries the majority of the point mutations that confer familial prion disease. Investigation of mutant PrPs finds a systematic relationship between the type of mutation and the apparent strength of this domain structure. The structural features identified here suggest mechanisms by which physiologic metal ions trigger PrP(C) trafficking and control prion disease.

Figure 1. Sequence and Structure of Human PrP^C

a) Schematic diagram of PrP (human sequence) with numbering indicating secondary structure, post-translational modifications, and pathogenic/ protective mutations. The N-terminus, which contains the octarepeat domain, is unstructured in the absence of Zn²⁺. The structured C-terminal domain possesses two N-linked carbohydrates (ovals), a disulfide bond, and a GPI anchor. The inherited pathogenic mutations that involve amino acid substitutions resulting in a change of charge are indicated in red. The protective inherited mutation is in blue. b) Zn²⁺ binds to the histidines within the octarepeat domain, as shown in the preliminary three-dimensional model.

Figure 2. Selected region of the ¹H-¹⁵N HSQC NMR spectra of wild type MoPrP

The black spectrum is from the protein in the absence of Zn²⁺. The red spectrum is from addition of 3 equivalents of ZnCl₂ and shows chemical shift changes, and loss or broadening of select cross peaks.

Figure 3. Zn²⁺ affects specific C-terminal residues

a) Ribbon diagrams showing residues that correspond to the disappeared (black) and significantly shifted (dark gray) peaks in the wild type MoPrP. b) The corresponding surface diagram for the full-length, wildtype MoPrP and c) the C-terminal construct MoPrP(91–230), lacking the Zn²⁺ binding N-terminal octarepeat domain.

Figure 4. Zn²⁺-mediated inter-domain interaction measured by DEER

a) Schematic representation of MoPrP showing the location of spin labeled side chains in relation to known secondary structures. b) Background -corrected dipolar evolution spectra and distance distributions obtained by Tikonov regularization for the three doubly-labeled constructs in the absence of Zn²⁺ (blue) and in the presence of 2mM ZnCl₂ (red). c) Dipolar evolution spectra and distance distributions for MoPrP Q66R1/ T200K1 with 0, 100, and 500µM ZnCl². Protein concentrations were between 80 and 100 µM.

Figure 5. C-Terminal mutations that affect the course of prion disease show different interaction patterns from wild type

Surface diagrams showing the disappeared (black) and significantly shifted (dark gray) peaks in the path ogenic mutants (a–d), and the dominant negative mutant (e).

Figure 6. Normalized peak intensities show different patterns for disease mutants, relative to wild type and the dominant negative

a) Cross peak intensity profiles for helices 1, 2 and 3 are compared for all proteins in this study. Asterisks mark unassigned peaks and the dashed lines represent the average I/ I_o values. b) A plot of the average I/ I_o for each helix comparing wild type and protective mutant (black squares) with pathogenic mutant s (gray circles). Residues in helix 2 exhibit lower I/ I_o (greater broadening) for wild type and dominant negative compared to familial mutants.

Figure 7. Electrostatic surface diagrams of wild type MoPrP and mutants

a) wild type as compared to the pathogenic mutants in b) and c). d) is the dominant negative. Blue denotes positive charge and red is negative charge.

Figure 8. Model of the Zn²⁺ mediated N-terminal–C-terminal interaction

The octarepeat domain (purple) containing the bound Zn²⁺ (red) was docked against the relevant C-terminal surface using restraints from DEER and NMR. The acidic residues that define the negative patch on helices 2 and 3 are highlighted.