Engineered sequestrins inhibit aggregation of pathogenic alpha-synuclein mutants

Abstract

Misfolding and aggregation of the neuronal protein alpha-synuclein (aSyn) has been identified as a hallmark of Parkinson's disease (PD) pathology and other synucleinopathies. Preventing formation of intracellular aSyn accumulations constitutes a therapeutic strategy against disease development. We recently reported on a new type of affinity protein, denoted Sequestrin, aimed for efficient and stable interactions with aggregation-prone amyloidogenic proteins and peptides. Upon binding, sequestrins interact with the aggregation-prone peptide and form a stabilizing four-stranded beta sheet with similarities to the beta sheet rich structures seen in amyloid fibrils. Here, high-affinity aSyn-binding sequestrins were isolated from a large naïve sequestrin library using phage display technology. The best binders demonstrated dissociation constant, K_D, values in the 10 nM-range, and structural rearrangements in both the sequestrin and aSyn protein upon binding. Modelling using AlphaFold, followed by NMR spectroscopy suggested that the sequestrins bind an N-terminal region of aSyn that is critical for amyloidogenic aggregation. In an in vitro aggregation study, the sequestrins demonstrated complete inhibition of aSyn aggregation at equimolar concentrations, including the three familial mutants A30P, E46K, and A53T that are associated with Parkinson's disease and Lewy body dementia.

Keywords: Parkinson’s disease; affibody molecule; alpha-synuclein; directed evolution; phage display; sequestrin.

Figure 1

Schematic overview of the sequestrin library and phage-display selected sequences against aSyn_1-140. (A) Illustration of the sequestrin (Sq) library with the two domains genetically connected by a flexible linker (grey), a disulphide bridge (yellow) and highlighting the eleven randomized positions in each domain (purple). The structure is based on PDB entry 2OKT and using the Alzheimer´s-associated amyloid beta (Aβ_1-40) peptide (white-transparent) as a model peptide. (B) The amino acid sequence of the sequestrin library (Sq_LIB) and ten phage-display selected candidates towards aSyn. Randomized residues are shown as “X” (purple).

Figure 2

Surface plasmon resonance (SPR) sensorgrams showing the interaction of aSyn_1-140, captured on a sensor chip, and four sequestrins Sq_aSyn2 (A), Sq_aSyn3 (B), Sq_aSyn4 (C) and Sq_aSyn11 (D), respectively, at 37°C. Grey curves are referenced raw data and black lines are fitted curves. All analytes were run in duplicate at concentrations from 3000 nM to 375 nM in dilution series. Equilibrium dissociation constants for the interaction between each sequestrin and aSyn were estimated using a 1.1 Langmuir model fit.

Figure 3

Circular dichroism (CD) spectroscopy characterization, including secondary structure components and variable temperature measurements (VTM), of four Sq_aSyn:aSyn_1–140 complexes. (A-D) Secondary structure content of free aSyn_1-140 (yellow), free sequestrins (green), Sq_aSyn:aSyn complexes at equimolar concentrations (blue), and the structure that was lost upon binding (red), i.e. the calculated difference between the sum of the free spectra and the complex spectrum. (E-H) Variable temperature measurements (VTM) between 20-90°C at 221 nm for the sequestrins (blue) or in complex with 15.7 μM molar equivalents of aSyn (black). (I-L) CD spectra for sequestrins co-incubated with molar equivalents of aSyn before (blue) and after (black) heat-induced denaturation.

Figure 4

Structure analysis of the aSyn: Sq_aSyn4 complex by in silico modelling and NMR spectroscopy. (A, B) NMR spectroscopy of the Sq_aSyn4:aSyn_1–140 complex. (A) ¹H-¹⁵N HSQC spectrum of uniformly ¹⁵N-labelled aSyn_1–140 in absence (black) and presence (red) of 1:1 molar equivalent of Sq_aSyn4. (B) Relative changes in peak intensities along the sequence of aSyn_1–140 upon addition of Sq_aSyn4 in molar aSyn: Sq_aSyn4 ratios from 6:1 to 1:1. (C, D) Structure prediction by AlphaFold3, illustrating the interaction of alpha synuclein and Sq_aSyn4 from two different perspectives. The model is based on a short segment of aSyn comprising the β-hairpin forming N-terminal region (red) and Sq_aSyn4 (light blue). Confidence metrics for the model were pTM 0.76, ipTM 0.73.

Figure 5

Aggregation time course of aSyn_1–140 wt or the familial mutants A30P, E46K, and A53T in the absence (orange) or presence of 1:1 (green) or 1:5 (blue) or 1:10 (purple) molar equivalents of the four sequestrins (A-D) Sq_aSyn2, (E-H) Sq_aSyn3, (I-L) Sq_aSyn4, (M-P) Sq_aSyn11. Aggregation was monitored by thioflavin T fluorescence for 72 h, and is presented as mean ± SD.