Prion protein-detergent micelle interactions studied by NMR in solution

Abstract

Cellular prion proteins, PrP(C), carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90-231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90-231) and mPrP[M129V]-(91-231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102-127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102-127 affect the interactions of PrP(C) with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.

FIGURE 1.

Detergent and proteins used in this study. A, zwitterionic form of DPC. B, schematic diagram of the mPrP-(90–231) polypeptide indicating the locations of the regular secondary structures, i.e. three α-helices and two strands of an antiparallel β-sheet, a “positively charged cluster” (CC) of amino acid residues in positions 95–111, and a “hydrophobic polypeptide segment” (HPS) comprising residues 112–127. C, amino acid sequence alignment of residues 90–135 for wild-type mPrP-(90–231) and the protein variants studied in this paper, where for each variant mPrP the amino acid replacements are given and identical residues are indicated by dots; the numbering is according to Schätzl et al. (27).

FIGURE 2.

Protein two-dimensional ¹⁵N,¹H-HSQC spectra recorded in the presence of 12.9 mm DPC (red contours) or without addition of DPC (blue contours). Backbone ¹⁵N-¹H resonances of selected residues in the hydrophobic polypeptide segment (see Fig. 1) are labeled with the one-letter amino acid code and the positions in the sequence. The dotted lines connect corresponding peaks in the spectra recorded in the presence and absence of DPC. A, mPrP-(90–231); B, mPrP[P105L]-(91–231); and C, mPrP[A117V]-(90–231).

FIGURE 3.

Histograms of the ¹⁵N chemical shift changes induced by the addition of 6.8 mm DPC for wild-type mPrP-(90–231) and eight variants thereof. The data are plotted versus the residue numbers. At the top, the locations of the regular secondary structure elements in mPrP-(90–231) are shown. Δδ(¹⁵N) is the difference between the ¹⁵N chemical shifts in the absence of DPC and in the presence of 6.8 mm DPC. The locations of the amino acid substitutions in the variant proteins are indicated by asterisks. A, mPrP-(90–231); B, mPrP[M129V]-(91–231); C, mPrP[P102L]-(91–231); D, mPrP[P105L]-(91–231); E, mPrP[P105L,M129V]-(91–231); F, mPrP[A117V]-(90–231); G, mPrP[A117V,M129V]-(90–231); H, mPrP[A113V,A115V,A118V]-(90–231); I, mPrP[K110I,H110I]-(90–231).

FIGURE 4.

Dependence of protein ¹⁵N chemical shifts on the DPC concentration for selected residues in mPrP[A113V,A115V,A118V]-(90–231) that are identified in the upper left corner. Δδ(¹⁵N) is the difference between the ¹⁵N chemical shifts in the absence of DPC and in the presence of the DPC concentrations given on the horizontal axis. The dashed vertical line indicates the cmc of DPC.

FIGURE 5.

Dependence of protein ¹⁵N chemical shifts on the DPC concentration. Same presentation as in Fig. 4, but for DPC concentrations above the cmc. The residues studied in all the experiments are identified in panel A. A, mPrP-(90–231); B, mPrP[M129V]-(91–231); C, mPrP[P102L]-(91–231); D, mPrP[P105L]-(91–231); E, mPrP[P105L,M129V]-(91–231); F, mPrP[A117V]-(90–231); G, mPrP[A117V,M129V]-(90–231); H, mPrP[A113V,A115V,A118V]-(90–231); and I, mPrP[K110I,H110I]-(90–231).

FIGURE 6.

Histograms of ¹³C^α chemical shift changes in mPrP-(90–231) and two variants thereof between solutions containing, respectively, 12.9 mm DPC and no DPC. The data are plotted versus the residue numbers. At the top, the locations of the regular secondary structures in mPrP-(90–231) are indicated. Δδ(¹³C^α) is the difference between the chemical shifts of the α-carbons observed at 12.9 mm DPC and in the absence of DPC. The locations of the amino acid substitutions in the prion protein variants are indicated by asterisks. A, mPrP-(90–231); B, mPrP[P105L]-(91–231); and C, mPrP[A117V]-(90–231).