The role of disulfide bond in the amyloidogenic state of beta(2)-microglobulin studied by heteronuclear NMR

Abstract

beta(2)-Microglobulin (beta2-m) is a major component of dialysis-related amyloid fibrils. Although recombinant beta2-m forms needle-like fibrils by in vitro extension reaction at pH 2.5, reduced beta2-m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of beta2-m in the acid-denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. [(1)H]-(15)N NOE at the regions between the two cysteine residues (Cys25-Cys80) revealed a marked difference in the pico- and nanosecond time scale dynamics between that the acid-denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, DeltaCalpha, DeltaCO, and DeltaHalpha, and (3)J(HNHalpha) coupling constants indicated that both the oxidized and reduced beta2-m at pH 2.5 have marginal alpha-helical propensity at regions close to the C-terminal cysteine, although it is a beta-sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of beta2-m, and that the marginal helical propensity at the C-terminal regions might play a role in modifying this potential.

Fig. 1.

Amino acid sequence (A) and schematic structure (B) of β2-m. Secondary structures are indicated with hydrogen bonds (A) and the numbering of β-strands (A,B). The locations of strongly (red) and weakly (blue) protected amide protons are indicated. The figure was produced using MOLMOL (Koradi et al. 1996) with the structure (PDB entry 3HLA) reported by Bjorkman et al. (1987).

Fig. 1.

Amino acid sequence (A) and schematic structure (B) of β2-m. Secondary structures are indicated with hydrogen bonds (A) and the numbering of β-strands (A,B). The locations of strongly (red) and weakly (blue) protected amide protons are indicated. The figure was produced using MOLMOL (Koradi et al. 1996) with the structure (PDB entry 3HLA) reported by Bjorkman et al. (1987).

Fig. 2.

AFM images of amyloid fibrils and filaments of β2-m. (A–C) Intact β2-m fibrils; (D–F) reduced β2-m filaments. (A,B) Amplitude images; (C–E) topographic images; (F) a 3D image. The full scales of (A) and (D) are 5 μm and those of (B), (C), (E), and (F) are 2 μm. The scale on the bottom represents the height of pixels in the image; the lighter the color the higher the feature is from the surface.

Fig. 3.

(Panel C appears on facing page.) ¹H-¹⁵N HSQC spectra of β2-m in the native state at pH 6.5 (A), the acid-denatured states of intact β2-m (B), and reduced β2-m (C) at pH 2.5. Temperature was 37°C. The underlined residues had weak peak intensities.

Fig. 3.

(Panel C appears on facing page.) ¹H-¹⁵N HSQC spectra of β2-m in the native state at pH 6.5 (A), the acid-denatured states of intact β2-m (B), and reduced β2-m (C) at pH 2.5. Temperature was 37°C. The underlined residues had weak peak intensities.

Fig. 4.

Secondary chemical shifts of backbone resonances versus residue number of β2-m in the native state at pH 6.5. (A) ΔCα, (B) ΔCβ, and (C) ΔHα. The locations of secondary structures obtained from the X-ray structure and Cys residues are indicated. The locations of highly (more than 24 h) and moderately (at least 10 min) protected amide protons are indicated by closed and open bars, respectively, under the locations of secondary structures. The chemical shift of the random coil was calculated on the basis of the previous report by Wishart et al. (1995).

Fig. 5.

Secondary chemical shifts of backbone resonances versus residue number of the acid-denatured β2-m at pH 2.5. (A,B) ΔCα, (C,D) ΔCO, (E,F) ΔHα of the intact β2-m (A,C,E) and reduced (B,D,F) β-2m. (G) ³J_HNHα values for the acid-denatured state of intact β2-m. (H) Helical propensity prediction by the program AGADIR (Múnos and Serrano 1994). The locations of secondary structures obtained from the X-ray structure are indicated in (A) and (B). For ΔCα and ΔCO, correction factors reported by Schwarzinger et al. (2001) were taken into consideration.

Fig. 6.

{¹H}-¹⁵N NOE heteronuclear NOE for the backbone amide nitrogen atoms of the intact (A) and reduced (B) β2-m measured at 500 MHz on the normal scale at pH 2.5 and 37°C. (C) Hydrophobicity of β2-m plotted against residue number. Hydrophobicity was calculated using the scale of Kyte and Doolittle (1982) averaged over a sliding window of seven residues. The locations of secondary structures obtained from the X-ray structure and Cys residues are indicated in (A).