Impact of N-Terminal Acetylation of α-Synuclein on Its Random Coil and Lipid Binding Properties

Abstract

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson’s disease, is common in mammals. The impact of this modification on the protein’s structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone 3JHH couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and 3JHH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an “initiation–elongation” process of binding to the membrane.

Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

“…Assuming that the low-abundance lipids PS and PI are sequestered in the inner cavity of CcO, lysine residues in this region might undergo electrostatic interactions with the headgroups of the lipids. Acetylation of these amino groups would thus modulate the stability of dimeric CcO, in line with previous reports of lysine acetylation changing lipid binding properties (43). PTM clusters at protein interfaces and in unstructured regions have been described previously; moreover, their importance for stabilizing and controlling protein interactions has been proposed (44).…”

N-terminal Acetylation Stabilizes N-terminal Helicity in Lipid- and Micelle-bound α-Synuclein and Increases Its Affinity for Physiological Membranes

“…We and others have shown that the primary consequence of N-terminal acetylation in the free state of aSyn is to increase the helicity of the N-terminal ϳ10 residues (30,32,33), an effect that likely results from the known ability of an N-acetyl group to act as a helix cap (71,72), which would stabilize the transiently helical structure formed at the N terminus. We (35,39) and others (33,73) have postulated that transient helical character at the very N-terminal region may be important in a The population of all bound states was calculated as the ratio of the average intensity ratio of residues 3-9, which are expected to be bound in both fully and partly helical binding modes, to the average intensity ratio of residues 129 -137, which remain unbound even at high lipid concentrations, subtracted from 1. b The population of the extended helix state was calculated as the ratio of the average intensity ratio of residues 65-80, which are in the second half of the lipid binding domain and have well resolved HSQC peaks, to the average intensity ratio of residues 129 -137, subtracted from 1. c Apparent dissociation constants were derived from fitting the bound populations of each state at several lipid concentrations to Equation 2, derived from a simple bimolecular binding equilibrium. See "Experimental Procedures" for a further description.…”

“…Prior studies of the effects of N-terminal acetylation on membrane binding by aSyn reported conflicting results, with one indicating an absence of any effect (30), whereas the other indicated a significant increase in binding (33). The first study employed large unilamellar vesicles with very high negative charge content, whereas the second study utilized small unilamellar vesicles with moderate negative charge content, but neither study investigated the influence of differing curvatures or charge content.…”

“…Nevertheless, N-terminal acetylation does influence the secondary structure of the protein (30,32,33). Because synuclein undergoes a disorder-to-helix transition upon binding to membranes and membrane mimetics (34,35), it might be expected that the effects of N-terminal acetylation on helical secondary structure could alter aSyn-membrane interactions.…”

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

“…[22][23][24] Experiments have yielded valuable results on many aspects of IDP structure and function. The most powerful methods are single molecule FRET experiments, which can provide information on the overall dimensions 25 and dynamics 26 of unbound IDPs, and NMR experiments which give more localised and higher resolution structural data 27,28 and also mechanistic insights. 29 Kinetic experiments can yield information on binding mechanism, [30][31][32][33][34][35] in particular when used in conjunction with a protein engineering approach.…”