The N-Terminus of the Intrinsically Disordered Protein α-Synuclein Triggers Membrane Binding and Helix Folding

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein ␣-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.

Section: As Labeling and Labelsupporting

confidence: 84%

mentioning

confidence: 99%

Specificity and Kinetics of α-Synuclein Binding to Model Membranes Determined with Fluorescent Excited State Intramolecular Proton Transfer (ESIPT) Probe

Shvadchak¹,

Falomir-Lockhart²,

Yushchenko³

et al. 2011

“…It is clear that N-terminal acetylation enhances the helicity of aSyn at its N terminus, and because membrane-bound aSyn becomes highly helical, it is reasonable that N-terminal acetylation would increase the affinity of the protein for membranes. Indeed, we and others have proposed previously that helicity at the N terminus of synuclein is likely a key mediator of early lipid interactions (35,73). On the other hand, N-terminal acetylation eliminates the primary amine group of aSyn and thereby decreases the positive charge of its N-terminal lipid binding domain by 1 at physiological pH.…”

Section: Discussionmentioning

confidence: 84%

“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Dikiy

Eliezer

2014

168

156

Background:The functional effects of normal N-terminal acetylation of the Parkinson disease protein ␣-synuclein are unknown. Results: N-Acetylation stabilizes helical structure at the N terminus of membrane-bound forms of synuclein, including a novel partly helical state. Conclusion: Stabilization of helicity increases affinity for membranes similar to synaptic vesicles. Significance: In vivo N-acetylation of ␣-synuclein likely affects its physiological function and dysfunction.

“…Moreover, on SUVs, multiple ␣S-binding modes (i.e. structural inhomogeneity) exist (30), in accordance with an inhomogeneous sequence distribution of surfactant affinities (29,31). Thus, although an elongated helix will dominate vesicle-bound ␣S, it is unclear to what extent broken helix conformations are populated on SUVs resembling synaptic vesicles.…”

mentioning

confidence: 99%

α-Synuclein Populates Both Elongated and Broken Helix States on Small Unilamellar Vesicles

Lokappa

Ulmer

2011

The misfolding of the protein ␣-synuclein (␣S) has been implicated in the molecular chain of events leading to Parkinson disease. Physiologically, ␣S undergoes a transition from a random coil to helical conformation upon encountering synaptic vesicle membranes. On analogous small unilamellar vesicles (SUVs), the conformation of ␣S is dominated by a single elongated ␣S helix. However, alternative broken helix states have been postulated, mandating experimental clarification. Here, the upper limit for the free energy difference between elongated and broken helix conformations on SUVs resembling synaptic vesicles was determined to be 1.2 ؎ 0.4 kcal/mol, which amounts to a population ratio of 7.6:1 between both states (12% broken helices). In response to helix breaks at different positions, ␣S rearranged in an opportunistic manner, thereby minimizing helix abrogations to as little as one to two turns. Enthalpy and entropy measurements of gel state SUV-␣S interactions indicated that broken helix states retain the ability to relieve membrane-packing stress. Thus, broken helix states are a distinct physiological feature of the vesicle-bound ␣S state, making it a "checkered" protein of multiple parallel conformations. A continuous interconversion between structural states may contribute to pathological ␣S misfolding. The protein ␣-synuclein (␣S)2 was discovered in amyloid plaques and as a gene whose expression is altered during the period of song learning in the zebra finch (1, 2), well representing subsequent discoveries regarding the pathological and physiological nature of this protein. In humans, each of the point mutations A30P, E46K, and A53T, as well as gene triplication, gives rise to familial parkinsonism and dementia (3-6). In conjunction with the propensity of ␣S to misfold into amyloid fibrils in vitro and in vivo (7,8), this places ␣S at the center of molecular events leading to prevalent human neurodegenerative disorders such as Parkinson disease (9). Physiologically, ␣S is a presynaptic brain protein that co-localizes with synaptic vesicles (10, 11). The binding and concomitant stabilization of such vesicles may modulate the threshold of neurotransmitter release, i.e. neural plasticity (1, 12, 13). In addition, ␣S can promote SNARE (soluble NSF attachment protein receptor) complex assembly (14).In aqueous solution, ␣S is highly soluble yet lacks secondary structure (15). It may therefore be considered to be an intrinsically unfolded protein. Based on the repetitive amphiphilic amino acid sequence of its N-terminal 89 residues (see Fig. 1A), it was early on proposed and verified that ␣S adopts predominantly helical conformation upon associating with small unilamellar vesicles (SUVs) (1, 16). Recently, the average structure of ␣S when bound to SUVs composed of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-snglycero-3-phospho-L-serine (POPS) lipids was reported (17). This structure, which was based on EPR distance and membrane immersion measurements, assigned an uninterrupted el...