Dynamics of a truncated prion protein, PrP(113-231), from (15)N NMR relaxation: order parameters calculated and slow conformational fluctuations localized to a distinct region

Abstract

Prion diseases are associated with the misfolding of the prion protein (PrP(C)) from a largely alpha-helical isoform to a beta-sheet rich oligomer (PrP(Sc)). Flexibility of the polypeptide could contribute to the ability of PrP(C) to undergo the conformational rearrangement during PrP(C)-PrP(Sc) interactions, which then leads to the misfolded isoform. We have therefore examined the molecular motions of mouse PrP(C), residues 113-231, in solution, using (15)N NMR relaxation measurements. A truncated fragment has been used to eliminate the effect of the 90-residue unstructured tail of PrP(C) so the dynamics of the structured domain can be studied in isolation. (15)N longitudinal (T(1)) and transverse relaxation (T(2)) times as well as the proton-nitrogen nuclear Overhauser effects have been used to calculate the spectral density at three frequencies, 0, omega(N,) and 0.87omega(H). Spectral densities at each residue indicate various time-scale motions of the main-chain. Even within the structured domain of PrP(C), a diverse range of motions are observed. We find that removal of the tail increases T(2) relaxation times significantly indicating that the tail is responsible for shortening of T(2) times in full-length PrP(C). The truncated fragment of PrP has facilitated the determination of meaningful order parameters (S(2)) from the relaxation data and shows for the first time that all three helices in PrP(C) have similar rigidity. Slow conformational fluctuations of mouse PrP(C) are localized to a distinct region that involves residues 171 and 172. Interestingly, residues 170-175 have been identified as a segment within PrP that will form a steric zipper, believed to be the fundamental amyloid unit. The flexibility within these residues could facilitate the PrP(C)-PrP(Sc) recognition process during fibril elongation.

Figure 1

Series of strips from ¹H-¹H_N slices of the 3D ¹⁵N NOESY-HSQC collected at 600 MHz (¹H) and 30°C. Each strip corresponds to a single residue in the two-stranded β-sheet, β1 (G125–S131), and β2 (Q159–R163). Solid lines connect sequential residues, dashed lines highlight connections occurring between the strands. The assignments are shown at the top of each strip.

Figure 2

¹⁵N HSQC assignments for mPrP(113–231). (a). Mouse PrP sequence of the C-terminal domain used in this study. (b) Annotated ¹⁵N HSQC spectrum. Numbering relates to the sequence in (a). The C-terminal His-tag (232–239) is unassigned. Dashed lines connect side chains of unassigned Q and N sidechains. Boxes highlight folded arginine side-chain amine groups (w₁ = w_1,app – 30 ppm). The “indole” peak is from Trp 144. The A and S are Ser and Ala residues at the N-and C-terminus.

Figure 3

Relaxation data for mPrP(113–231). (a) Heteronuclear NOE and the relaxation rates, (b)R₁ (=1/T₁), and (c)R₂ (=1/T₂) determined at 600 MHz. Sample conditions: 30°C; pH 5.3; 20 mM acetate buffer.

Figure 4

Reduced spectral density functions: (a) J(0), (b) J(ω_N), and (c) J(0.87ω_H) for mPrP(113–231) at 600 MHz. The high frequency spectral densities, J(0.87ω_H), indicate fast pico-second motions typically observed in flexible regions of proteins. J(0) indicates sub-nanosecond flexibility of the NH bond vector, the smaller the value of J(0) the greater the flexibility. Uncharacteristically large J(0) indicate residues with slow conformational fluctuations. Sample conditions: 30°C; pH 5.3; 20 mM acetate buffer.

Figure 5

(a) Order parameters (S²) for mPrP(113–231). Order parameters describe the amplitude range of the nanosecond time-scale motions between 0 (flexible) and 1.0 (rigid). Residues that exhibit additional R_ex exchange motions are highlighted in red. (b) Order parameter mapped onto the structure (1 xyx) of mPrP^C: red S² > 1.0 (R_ex motions); blue 0.85 < S² < 1.0; cyan 0.75 < S² < 0.85; green 0.65 < S² < 0.75; and yellow S² < 0.65, residues in gray have no value calculated. (c) Slow conformational fluctuations are localized to a distinct region within PrP^C, the position of side-chains that exhibit R_ex motions are highlighted, dots are proton positions. (d) The molecular surface, in red, generated by the residues that exhibit R_ex motions, within the context of the whole structured domain. This figure was generated using GRASP2.