Molecular basis for preventing α-synuclein aggregation by a molecular tweezer

Abstract

Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.

Keywords: Inhibitor; Intramolecular Diffusion; Mass Spectrometry (MS); Parkinson's Disease; Protein Aggregation; Spectroscopy; α-Synuclein.

FIGURE 1.

Schematic structures of α-synuclein and CLR01. A, in the α-synuclein sequence, the Ala-69→Cys and Tyr-94→Trp substitutions are shown in red. The Lys residues are marked by green arrows and boldface as possible binding sites for CLR01. The identified putative binding sites at Lys-10 and Lys-12 are highlighted in yellow. B, diagram of CLR01 bound to a lysine side-chain.

FIGURE 2.

Effect of CLR01 binding on Trp-94 fluorescence and CLR01 fluorescence. A, fluorescence spectra of 1 μm α-synuclein in the presence of increasing concentrations of CLR01. B, total fluorescence between 300–450 nm versus [CLR01]. The line is the fit of the first seven points to a hyperbola. C, first (black) and second (red) most significant spectra from single value decomposition of all spectra. D, corresponding amplitudes of each spectrum versus [CLR01]. The black lines are sigmoidal fits to sections of the data.

FIGURE 3.

Binding sites of CLR01 on α-synuclein investigated by ECD-MS/MS. A, ESI mass spectrum of 10-μm α-synuclein and 10-μm CLR01 shows 1:1 binding stoichiometry (singly charged unbound CLR01 was observed at m/z 744). B, schematic of the ECD-MS/MS fragmentation profile for the 13+-charged 1:1 α-synuclein:CLR01 complex. Protein fragments of the c- (retaining the N terminus) and z*- (retaining the C terminus) product-ion series were observed. Some product ions from dissociation of the polypeptide backbone corresponded to unbound peptide (black line), whereas other product ions retained binding to CLR01 (red line). At some positions along the peptide chain, product ions were observed in both CLR01-bound and unbound states. From the fragmentation profile, residues 10–20 that include Lys-10 and Lys-12 are suggested to be the site(s) of CLR01 binding.

FIGURE 4.

PICUP analysis of α-synuclein oligomerization. PICUP was performed on α-synuclein in the absence or presence of 1 or 10 molar equivalents of CLR01. a, following cross-linking, the products were fractionated by SDS-PAGE and visualized by silver staining. Positions of molecular weight markers are shown on the left. The gel is representative of each of three independent experiments done in triplicates. b, percent abundance of the three bands of the trimer and c, tetramer products for 30 μm protein. d and e, measured abundances of N-mers after PICUP for various ratios of CLR01:α-synuclein. The protein concentrations are (d) 3 μm and (e) 30 μm.

FIGURE 5.

Measurement of α-synuclein oligomerization by Trp fluorescence. a, 45 μm [Cys69,Trp94] α-synuclein were incubated at 37 °C with stirring in a) the absence, or b) the presence of equimolar CLR01 and Trp fluorescence was measured at various times points. The arrows indicate increasing time. c, maximum intensity versus time for various molar ratios of CLR01 (0:1, black points; 1:1, red points; 2:1, blue points). The lines are linear fits to the data.

FIGURE 6.

Measurement of Trp-94-triplet quenching. a, typical kinetic decay of the Trp triplet-state as measured by transient optical absorption. The data (black points) are fit to two exponential decays. The slower decay corresponds to various photophysical processes created by the UV pulse (e.g. solvated electrons), whereas the faster decay corresponds to intramolecular contact between Trp-94 and Cys-69. b, observed decay times versus solution viscosity for various temperatures. The lines are independent fits for each temperature. Reaction-limited (c) and diffusion-limited (d) rates of the Trp-Cys quenching measurement versus temperature for various molar ratios of CLR01:α-synuclein. The bars in c represent the lower limit of k_R as determined by the inverse error of the intercept from Equation 2. e, average root mean square distance between Trp-94 and Cys-69 determined for each reaction-limited rate using Equations 3 and 5. f, intramolecular diffusion coefficients determined for each diffusion-limited rate using Equation 4.

FIGURE 7.

Effect of CLR01 on α-synuclein oligomerization. a, reconfiguration rates at 30 °C versus molar ratio of CLR01:α-synuclein calculated as k_r = D/<r²>. The black line is a linear fit to the data. b, second-order formation rates of oligomer (O) calculated from the model in Reaction Scheme 1 using rates as noted in the text. The black line is a fit to a hyperbola.

FIGURE 8.

Calculated effect of CLR01 binding on α-synuclein conformation. a, re-weighted probability distributions, Z(r), for σ = 0, γ = 20 for α-synuclein alone (blue), α-synuclein with one CLR01 bound at Lys-10 (cyan), α-synuclein with one CLR01 bound at Lys-12 (magenta), and α-synuclein bound to two CLR01 molecules in positions 10 and 12 (red). In each case, the bound CLR01 are approximated by Lys→Glu substitutions. b, calculated (red triangles) reaction-limited rates using Equation 9 and the probability distributions calculated in a. The measured reaction-limited rates are plotted as black circles.