Covalent structural changes in unfolded GroES that lead to amyloid fibril formation detected by NMR: insight into intrinsically disordered proteins

Abstract

Co-chaperonin GroES from Escherichia coli works with chaperonin GroEL to mediate the folding reactions of various proteins. However, under specific conditions, i.e. the completely disordered state in guanidine hydrochloride, this molecular chaperone forms amyloid fibrils similar to those observed in various neurodegenerative diseases. Thus, this is a good model system to understand the amyloid fibril formation mechanism of intrinsically disordered proteins. Here, we identified a critical intermediate of GroES in the early stages of this fibril formation using NMR and mass spectroscopy measurements. A covalent rearrangement of the polypeptide bond at Asn(45)-Gly(46) and/or Asn(51)-Gly(52) that eventually yield β-aspartic acids via deamidation of asparagine was observed to precede fibril formation. Mutation of these asparagines to alanines resulted in delayed nucleus formation. Our results indicate that peptide bond rearrangement at Asn-Gly enhances the formation of GroES amyloid fibrils. The finding provides a novel insight into the structural process of amyloid fibril formation from a disordered state, which may be applicable to intrinsically disordered proteins in general.

FIGURE 1.

Structural characteristics of GroES. A, top view of the molecular structure of GroES heptamer, drawn using MOLMOL (60) with coordinates from PDB file 1AON. One subunit is drawn as a space-filling model. B, amino acid sequence of GroES. Thick arrows above the sequence indicate regions corresponding to β-strands seen in the native x-ray structure. The core sequence of the amyloid fibril of GroES determined previously (20) is boxed and shaded in gray.

FIGURE 2.

NMR peak assignments of GroES in the unfolded state. A, two-dimensional ¹H-¹⁵N HSQC spectrum of GroES. Assignments are indicated in the figure. Measurement conditions are: 10 mg/ml GroES, 10 mm sodium phosphate buffer, 1.6 m Gdn-HCl, 10% (v/v) D₂O (pH 6.5) at 25 °C. B, SSP of GroES. Chemical shifts of Cα and Cβ of GroES were used to calculate the residue-specific SSP scores. A SSP score of 1 or −1 at a given residue position reflects a fully formed α- or β-structure, respectively, and 0 reflects a random coil.

FIGURE 3.

Changes in NMR spectra of GroES after prolonged incubation at 25 °C. A, ¹H-¹⁵N HSQC spectra after a 2-day (black) and 28-day (red) incubation in 1.6 m Gdn-HCl without agitation at 25 °C. Notable changes: peak intensities of Asn⁴⁵, Gly⁴⁶, Asn⁵¹, and Gly⁵² decreased significantly, and the peaks of Gly⁴⁶ and Gly⁵² were detected partially at new positions (dotted square), indicating that intermediate species were formed. B, relative peak intensity of resonances at 2 days (black circles), 14 days (blue circles), and 28 days (red circles), given as δI = I/I₀, where I represents the peak intensities at 14 or 28 days and I₀ is equal to the intensity at 2 days. C–E, ¹⁵N relaxation parameters of GroES. The ¹H-¹⁵N NOE (C), relaxation data R₁ (D), and R₂ (E) of disordered species (black circles) and intermediate species (red triangles) are shown in each panel. The ¹⁵N relaxation parameters of Gly⁴⁶ and Gly⁵², detected at new positions in the intermediate species, are also plotted (red columns) along with those in the disordered species (black columns). The data of the disordered and intermediate species were recorded between day 0 and day 4, and between day 21 and 25, respectively.

FIGURE 4.

Separation of the intermediate species that are formed during GroES amyloid fibril formation. A, native-PAGE analysis of GroES native heptamer, GroES incubated in 1.6 m Gdn-HCl for 0–35 days, and mature GroES amyloid fibrils resolubilized in 7.5 m Gdn-HCl and refolded. Preparation of the incubated GroES in Gdn-HCl is as follows. Aliquots were taken during amyloid fibril formation (10 mg/ml GroES in 10 mm sodium phosphate buffer (pH 6.5), 1.6 m Gdn-HCl incubated without agitation at 25 °C in a Shigemi tube) and ultracentrifuged, and Gdn-HCl was removed from the supernatant using a PD Spin Trap G-25. After the removal of Gdn-HCl, refolding was allowed to occur. Mature amyloid fibrils were solubilized in 7.5 m Gdn-HCl, then Gdn-HCl was removed in similar fashion. Five micrograms of each sample were loaded onto each lane. In-gel digestion with lysyl endopeptidase and extraction of peptides from the relevant bands were performed followed by MALDI-TOF analysis. B–E, mass spectra of the target peptide Ser³⁵-Lys⁵⁵ (STRGEVLAVGNGRILENGEVK (M+H)⁺ is 2198.18) containing two Asn-Gly sequences shown for native heptamer (B) and intermediates I–IV marked in the native-PAGE gels (C–E), respectively. The parent ion mass value of the each spectrum is also shown in each panel.

FIGURE 5.

Amyloid fibril formation and the analysis of intermediates formed by various GroES mutants. A, amyloid fibril formation of GroES mutants monitored by Thioflavin-T fluorescence (1 mg/ml GroES, 10 mm sodium phosphate (pH 7.4), 1.6 m Gdn-HCl with linear agitation (90 min⁻¹) at 37 °C). WT, circles; N45A, squares; N51A, triangles; and N45A/N51A, diamonds. B, native-PAGE analysis of GroES native heptamer, incubated GroES (0, 24 h), and GroES mutants resolubilized and refolded from mature amyloid fibrils. The mass spectra of the target peptides of Ser³⁵-Lys⁵⁵ from GroES N45A and N51A (both (M+H)⁺ are 2155.18), which were derived from the bands i–iv in native-PAGE gel, are indicated in C–F, respectively. The parent ion mass of the each spectrum is also shown in each panel. G and H, ¹H-¹⁵N HSQC spectra taken of WT and N45A/N51A incubated for 28 days compared by overlaying ¹H slices corresponding to the region Gly⁴⁶ (G) and Gly⁵² (H) highlighted by the dotted square in Fig. 3A.

FIGURE 6.

Schematic model of the structural changes in early stage of GroES amyloid fibril formation. The region of Val⁴³-Leu⁵⁷ (denoted by a thick red line) has been identified as a region where structural changes occur early on during fibril formation. This region immediately precedes the fibril core region (Asp⁵⁸-Lys⁷⁴: denoted by blue arrows) and is apparently distinct from this region. This intermediate was monomeric, and the structural change was caused by the covalent rearrangement of Asn-Gly sequences. Especially, the formation of βAsp from Asn⁵¹ acts as a trigger for the following process. After formation of the intermediate species, a fibril nucleus may be formed fast at the adjacent core region, followed by very fast incorporation of disordered intact monomeric species (nucleus-dependent fibril extension). Therefore the mature amyloid fibrils of GroES contain both altered and unaltered molecular species. For details, see “Results” and “Discussion” sections.