De novo design of peptides that bind specific conformers of α-synuclein

Abstract

Insoluble amyloids rich in cross-β fibrils are observed in a number of neurodegenerative diseases. Depending on the clinicopathology, the amyloids can adopt distinct supramolecular assemblies, termed conformational strains. However, rapid methods to study amyloids in a conformationally specific manner are lacking. We introduce a novel computational method for de novo design of peptides that tile the surface of α-synuclein fibrils in a conformationally specific manner. Our method begins by identifying surfaces that are unique to the conformational strain of interest, which becomes a "target backbone" for the design of a peptide binder. Next, we interrogate structures in the PDB with high geometric complementarity to the target. Then, we identify secondary structural motifs that interact with this target backbone in a favorable, highly occurring geometry. This method produces monomeric helical motifs with a favorable geometry for interaction with the strands of the underlying amyloid. Each motif is then symmetrically replicated to form a monolayer that tiles the amyloid surface. Finally, amino acid sequences of the peptide binders are computed to provide a sequence with high geometric and physicochemical complementarity to the target amyloid. This method was applied to a conformational strain of α-synuclein fibrils, resulting in a peptide with high specificity for the target relative to other amyloids formed by α-synuclein, tau, or Aβ40. This designed peptide also markedly slowed the formation of α-synuclein amyloids. Overall, this method offers a new tool for examining conformational strains of amyloid proteins.

Fig. 1. Cryo-EM structures of WT (green) and E46K (cyan) αSyn fibrils. The most discriminating residues Gln62, Thr64, and Val66 project outward in PDB 6cu7 but inward in PDB 6ufr. E46 and K46 are buried in each structure.

Fig. 2. Amyloid peptide binder design pipeline. (A) Helix–strand designability is evaluated using MASTER and structural filters consisting of steric, contact, and secondary structure. This produced helix backbones favorable for helix–strand interaction. (B) Helix–helix designability is evaluated by applying the screw symmetry of amyloid structure on the helix and assessing their occurrence in the PDB using MASTER. (C) The peptide sequence was generated by Rosetta using the symmetry files, considering both interactions of helix–strand and helix–helix while fixing the amyloid residues.

Fig. 3. Raw confocal images of amyloids with FITC-labelled peptides 1*–4*. (A) WT αSyn fibril with peptides 1*–4*. (B) Peptide 4* with WT αSyn and E46K αSyn fibrils. (C) Peptide 4* with WT αSyn, Aβ40 and 0N3R tau fibrils. (D) Peptide 4* with three conformers of αSyn fibrils.

Fig. 4. (A) Docking prediction of the designs using MOE dock and Hdock. The backbone RMSD for design vs. prediction from MOE dock was 2.0 Å and 0.8 Å for Hdock. (B) MD simulation of design and the WT αSyn fibril (PDB 6cu7) with a plot showing the Cα RMSD calculation over 500 ns simulation.

Fig. 5. (A) Ligand depletion assay with peptide 4* reveals a saturation binding curve with K_d= 1.3 μM and binding stoichiometry of 1 peptide per 2 αSyn proteins (black). The scramble* control did not show appreciable binding (red) (B) ThT fluorescence assay with 300 μM αSyn monomer with or without 200 μM peptide 4. The mean fluorescence signal from octuplicate is shown in thicker line with standard deviation. A rapid increase in ThT fluorescence is observed at 500 min for αSyn alone whereas a slower increase is observed with the αSyn with peptide 4.