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. 2011 May 11;133(18):7152-8.
doi: 10.1021/ja2009554. Epub 2011 Apr 14.

Allostery in a disordered protein: oxidative modifications to α-synuclein act distally to regulate membrane binding

Eva Sevcsik  1 , Adam J Trexler, Joanna M Dunn, Elizabeth Rhoades
Affiliations

Allostery in a disordered protein: oxidative modifications to α-synuclein act distally to regulate membrane binding

Eva Sevcsik et al. J Am Chem Soc. .

Abstract

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson's disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first ∼95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with the lipid bilayer. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane-binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function.

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Figures

Figure 1
Figure 1
Schematic representation of aS showing modification sites. aS is divided into three regions: a positively charged N-terminal region, a hydrophobic central region (NAC region) forming the core of the β-sheet structures in amyloid fibrils, and an acidic C-terminus. The N-terminus and the NAC region form an amphipathic α-helix upon binding to lipid membranes. aS has four tyrosines which are indicated in black; the different modification sites for fluorescent labeling are indicated in gray.
Figure 2
Figure 2
Binding of aS to lipid vesicles. A. Normalized autocorrelation curves of aS with increasing lipid concentrations showing characteristic shift to longer decay times with increasing fraction of bound protein. B. Representative hyperbolic binding curves of aS (black) and nit-aS (gray) binding to 50 nm diameter 1:1 POPC/POPS vesicles at pH 7.4. Partition coefficients of the different aS constructs: (C) Y→F, unmodified (solid) and nitrated (diagonal hatch marks); and (D) Y→D nitration mimics.
Figure 3
Figure 3
Effect of nitration on conformation of aS in solution and bound to lipid vesicles. ETeff histograms of unmodified (rows 1 and 3, red fits) or nitrated (rows 2 and 4, blue fits) aS were measured in solution (upper two rows, dark gray) and in the presence of 100% POPS vesicles (lower two rows, light gray). In order to probes specific regions of the protein, aS was labeled at various positions: A–D, residues 9 and 54; E–H, residues 54 and 92; I–L, residues 72 and 115; M&N, residues 33 and 115. The peak at ETeff≈1 seen in some panels (for example, K and L) is the result of a trace contaminant present in some of the vesicle solutions. All histograms are normalized so that the area under the Gaussian fit curve (red or blue) equals 1.
Figure 4
Figure 4
Cartoon illustrating changes in aS conformation upon nitration and the effects upon membrane binding. The four constructs described in the Results section are marked with gray arrows; upon nitration, the arrows are color coded to indicate a measured change in distance: gray = no change; blue = increase; red= decrease.
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