Conformational switching in PolyGln amyloid fibrils resulting from a single amino acid insertion

Abstract

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of β-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-β structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10-20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated β-structure model for PolyQKd-33 and a model with two β-solenoid protofibrils for PolyQKd-32. These data provide evidence for β-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.

Figure 1

Schematic representation of the designed peptides. K, K_D, and S denote location of L-lysine, D-lysine, and L-serine, respectively. The remaining positions are occupied by L-glutamines, which are not denoted by letters, for greater clarity. The peptide shown in the scheme is PolyQKd-33. The peptide PolyQKd-32 has one less L-Gln in the middle section. The angles between β-strands indicated by dashed lines are arbitrary.

Figure 2

Images of in vitro-assembled PolyQKd-32 and PolyQKd-33 fibrils. Electron micrographs of negatively stained and vitrified PolyQKd-32 (A, B and E, F) and PolyQKd-33 fibrils (C, D and G, H), respectively with their corresponding averaged density profiles (arrows mark operationally defined edges). The scale bars shown in the density profiles are 5 nm. Scale bars indicate 100 nm in A, C, E, G and 10 nm in B, D, F, H.

Figure 3

Electron diffraction patterns and corresponding electron micrographs of unstained fibrils. (A) PolyQKd-33 fibrils and (B) PolyQKd-32 fibrils. The scale bar of 200 nm applies to both EM images. The ring in the diffractograms is at a spacing of 0.47 nm.

Figure 4

STEM images of in vitro assembled fibrils. STEM micrographs of (A) PolyQKd-32 and (B) PolyQKd-33 fibrils with clean background, typical of those used for mass-per-length measurements. Scale bar = 10 nm.

Figure 5

STEM microscopy and mass-per-length analysis. STEM micrographs of PolyQKd-32 (A) and PolyQKd-33 (B) fibrils with their corresponding transverse density profiles. Scale bar = 10 nm on micrographs and 5 nm on the density profiles (arrows mark operationally defined edges). Measurements were made only from uniform thin and apparently single-stranded regions, such as those delimited by white arrows. The regions marked by diamonds are thicker bundled fibril. Tobacco mosaic virus indicated by an asterisk was used as an internal calibration reference. (C) The distribution of PolyQKd-32 measurements has a mean at 1.54 ± 0.20 subunits per axial step (0.47nm), N = 780. (D) The distribution of PolyQKd-33 measurements has a mean at 1.10 ± 0.14 subunits per axial step, N = 816 (see also Fig. 4).

Figure 6

Models of amyloid fibrils built from three β-structured modules, each with three strands (arrows) and two turns. Lines between β-strands denote H-bonds. (A) β-meander model having three side-by-side β-sheets. Its width is between 2.7 and 3.7 nm and expected MPL is one molecule per cross section. In principle, β-meanders from the neighboring β-sheets can be axially displaced. (B) A β-solenoid model in a parallel conformation with estimated width of 1.8 to 3.7 nm and 2/3 molecule per fibril cross section. (C). A superpleated β-structure model in a parallel conformation with width of 2.9 to 3.7 nm and one molecule per fibril cross section.

Figure 7

Superpleated β-structure models of PolyQ amyloid fibrils built from three β-structured modules each with three strands (arrows) and two turns. Lines between β-strands denote H-bonds. The polyQ chains can be arranged in two antiparallel (left, and center) or a parallel manner (right). Molecular modeling favors antiparallel stacking type 2 (data not shown) where positively charged Lys is located at greater distances and this diminishes the electrostatic repulsions. Moreover, in the antiparallel arrangement type 1, β-arcs that are packed one over the other have steric tensions.

Figure 8

Ribbon representations (axial and lateral views) of structural models of PolyQKd-32 and PolyQKd-33 fibrils. Sequence motifs of β-arcs fit well blbbl conformation (41) (where b is β-conformation and l is left-handed α-conformation of residues). First, the D-lysine in the 10th and 21st positions of each β-arc should favor required left-handed α-conformation. Second, because uncompensated charges inside the protein strongly destabilize the structure, D-lysines in the 10th and 21st positions and L-lysines in the 13th and 24th positions should be on the surface of the engineered fibrils when the bends have the blbbl-arc. Third, for the inside positions of such β-arcs (positions 11, 14 and 22, 25) small serine was selected because this residue is frequently located there in the known blbbl-arcs. The modeled structures do not have steric tensions and donors and acceptors of H-bonds interact with each other or having the possibility to bind water molecules. In accordance with PROCHECK output, the overall average G-factor of the models are ∼0.50, a value typical for a good quality model.