Both Met(109) and Met(112) are utilized for Cu(II) coordination by the amyloidogenic fragment of the human prion protein at physiological pH

Abstract

The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five Cu(II) ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured "amyloidogenic" domain between residues 91 and 126 PrP(91-126). Herein we expand upon a previous study [J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710-719] where we demonstrated that the physiologically relevant high affinity Cu(II) coordination site within PrP(91-126) is found between residues 106 and 114. It was shown that Cu(II) is contained within a square planar (N/O)3S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the Cu(II) coordination environment within the PrP fragment containing residues 106-114 (PrP(106-114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106-114) with systematic Met-->Ile "mutations" we show that the CuII coordination environment within PrP(106-114) is actually comprised of a mixture of two major species; one Cu(II)(N/O)3S center with the M(109) thioether coordinated to CuII and another CuII(N/O)3S center with the M(112) thioether coordinated to CuII. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106-114) both reduces the CuII affinity of the peptide by two to seven fold, and renders the resulting CuII metallopeptides redox inactive. The biological implications of these findings are discussed.

Fig. 1

CD spectra obtained for Cu(M112I) (dotted spectrum), Cu(M109I) (dashed spectrum), and Cu(M109/112I) (solid spectrum) highlighting the ligand field. For comparison the CD spectrum of {Cu^II(PrP(106–114))} has been included (dotted and dashed spectrum). Inset: Depicts and expanded view of the CD spectrum Cu(M112I) (dotted spectrum), Cu(M109I) (dashed spectrum), Cu(M109/112I) (solid spectrum), and {Cu^II(PrP(106–114))} (dotted and dashed spectrum).

Fig. 2

Fluorescence intensity of the 350 nm W(99) emission from PrP(91–126) as a function of PrP(106–114)(M(nnn)I) concentration. The titration data for PrP(106–114)(Cu(M112I)) are given as circles, the data for PrP(106–114)(Cu(M109I)) are given as squares, and the data for PrP(106–114)(Cu(M109/112I)) are given as triangles. The closed, open, and dotted shapes depict individual trials. The lines (dotted: PrP(106–114)(Cu(M112I)); dashed: PrP(106–114)(Cu(M109I)); solid: PrP(106–114)(Cu(M109/112I))) depict the best fits to the titration data.

Fig. 3

A: Depicts the XANES region of the Cu K-edge X-ray absorption spectra of {Cu^II(PrP(106–114))} (dotted and dashed spectrum), Cu(M109I) (dashed), Cu(M112I) (dotted) and Cu(M109/112I) (solid). The metrical parameters refined for in the imidazole phase and amplitude function are also depicted in A. The magnitude FT k³ EXAFS and k³ EXAFS (insets) of Cu(M109I) (B), Cu(M112I) (C), and Cu(M109/112I) (D) are also depicted. The experimental data are depicted as the solid spectrum, the best fit to the data are the dashed spectrum, and the difference spectrum are the dotted spectrum.

Fig. 4

X-band EPR spectra of {Cu^II(PrP(106–114))}, Cu(M109I), Cu(M112I), and Cu(M109/112I) obtained at 20 K in 1:1 buffer:glycerol glasses (buffer = 50 mM NEM; pH 7.4).

Fig. 5

Simulation (thick solid spectrum) of the spectrum of {Cu^II(PrP(106–114))} (thick dotted-dashed spectrum) using a 5:3 ratio of the spectra of Cu(M112I) (thin dotted spectrum) and Cu(M109I) (thin dashed spectrum).

Fig. 6

The left hand figures depict the CD spectrum (top) and electronic absorption spectrum (bottom) of {Cu^II(PrP(106–114))} highlighting the S^Met → Cu(3d) transition. The right hand figures depict the calculated spectra for [Cu^II(KH)O]⁺ (A), [Cu^II(GKH)]⁺ (B), and [Cu^II(KH)N]⁺ (C). For the simulated spectra a line-width at half-height of 1000 cm⁻¹ was used for all transitions.

Fig. 7

Isosurface plots of the MOs for [Cu^II(KH)O]⁺ that make up the leading configuration for the ground states (A and B) and excited state (C) for the two S(σ)/Cu(3d)/N(σ)/O(σ) → Cu(3d_x²–y²)/S(σ)/N(σ)/O(σ)* transitions. The lysine side-chain has been removed for clarity.

Fig. 8

Alignment of various mammalian PrP primary protein sequences corresponding to PrP(91–126) from humans.

Chart 1

Proposed structure of Cu^II within the amyloidogenic PrP fragment of the human prion protein based on an X-ray absorption spectroscopic analysis.

Chart 2

DFT minimized structures of the computational metallopeptide models used in the study. Selected metrical parameters for these models are presented in Table 4.

Chart 3

Structures of [(pbnap)Cu-OMe]⁺ and [(L^SEP)Cu(H₂O)(OClO₃)]⁺.