Exploring the role of post-translational modifications in regulating α-synuclein interactions by studying the effects of phosphorylation on nanobody binding

Abstract

Intracellular deposits of α-synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post-translational modifications (PTMs) of α-synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C-terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post-mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α-synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C-terminal phosphorylation in modulating the interaction of α-synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C-terminal region of α-synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α-synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α-synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α-synuclein.

Keywords: Parkinson's disease; isothermal titration calorimetry; nanobody); nuclear magnetic resonance; phosphorylation; protein misfolding; single-domain antibody (sdAb; surface plasmon resonance; α-synuclein.

Figure 1

Schematic illustration of the sequence of α‐synuclein and its significance for the binding of NbSyn87. (A) Sequence of human α‐synuclein showing the seven imperfect repeats in the N‐terminal region, and the binding epitope of NbSyn87.21 The solid red line indicates the binding region of NbSyn87, while the dotted line marks the region experiencing resonance broadening in HSQC spectra that is indicative of a conformational change upon binding of the nanobody [Fig. S1(A), Supporting Information]. (B) The three distinct domains within the primary sequence of α‐synuclein are the N‐terminal domain, the NAC region, and the C‐terminal region. The arrows show the phosphorylation sites located within the C‐terminal region.

Figure 2

Interaction of NbSyn87 with α‐synuclein variants monitored by ITC. The top panels illustrate the ITC data for NbSyn87 binding to wild type and the S129A, S129E, pS129, and pY125 α‐synuclein variants at 25 °C in PBS buffer. The lower panels illustrate the integrated heat release at each titration point for each protein. The resulting binding isotherms were fitted to a 1:1 bimolecular binding model (see Materials and Methods section), and the values of the binding parameters are given in Table I.

Figure 3

Interaction of NbSyn87 with α‐synuclein variants monitored by SPR. (A) Triplicate kinetic traces for the binding of NbSyn87 (concentrations ranging from 227 to 0 nM) to immobilized wild type (WT), S129A, S129E, and pS129 α‐synuclein. The data are corrected for the signals originating from the buffer and for nonspecific binding to the dextran matrix surface and are fitted to a 1:1 binding isotherm (solid lines), enabling the k _on, k _off, and K _d values to be extracted for the interaction of NbSyn87 with each α‐synuclein variant. (B) A separate chip was prepared with immobilized wild type, S129A and pY125 α‐synuclein, in three different flow‐cells. Right panel: triplicate binding traces of NbSyn87 with immobilized pY125 α‐synuclein; Left panel: triplicate binding traces of NbSyn87 with S129A, immobilized in the flow‐cell adjacent to immobilized pY125 α‐synuclein. (C) Left panel: the equilibrium values of the binding traces shown in A. for the binding of NbSyn87 to wild type α‐synuclein and to the variants S129A, S129E, and pS129, as a function of the concentration of NbSyn87. Right panel: the equilibrium values of the binding traces shown in B. for the binding of NbSyn87 to pY125 α‐synuclein for concentrations ranging from 2500 to 0 nM (♦), for the binding of NbSyn87 to S129A α‐synuclein (□) for concentrations ranging from 625 to 0 nM, and for the binding of NbSyn87 to wild type α‐synuclein (▲), for concentrations ranging from 2500 to 0 nM.

Figure 4

NMR chemical shift changes observed for phosphorylated α‐synuclein variants. Chemical shift changes of phosphorylated (A) pY125 and (B) pS129 variants of α‐synuclein, calculated relative to the wild type protein. The chemical shift changes are defined as [0.04 × (δ₁₅N_{wild type} – δ₁₅N_variant)2 + (δ¹H_{wild type} – δ¹H_variant)2]^1/2.

Figure 5

¹H‐¹⁵N HSQC NMR spectra of phosphorylated α‐synuclein variants bound to NbSyn87. ¹H‐¹⁵N HSQC correlation spectra of uniformly ₁₅N labeled (A) wild type, (B) pS129, and (C) pY125 variants of α‐synuclein upon binding to NbSyn87, shown in blue, in comparison to those of the unbound form of each α‐synuclein variant, represented in red. The spectra of the bound proteins were measured in the presence of saturating concentrations of NbSyn87.