Comparison of Structure and Dynamics of Micelle-bound Human α-Synuclein and Parkinson Disease Variants

Abstract

Three point mutations (A30P, E46K, and A53T) as well as gene triplication genetically link the 140-residue protein ␣-synuclein (aS) to the development of Parkinson disease. Here, the structure and dynamics of micelle-bound aS(A30P) and aS(A53T) are described and compared with wild-type aS, in addition to describing the aS-micelle interaction. A53T is sensed only by directly adjacent residues and leaves the backbone structure and dynamics indistinguishable from the wild type. A30P interrupts one helix turn (Val 26 -Ala 29 ) and destabilizes the preceding one. A shift in helix register following A30P disturbs the canonical succession of polar and hydrophobic residues for at least two turns. The shortened helix-N adopts a slightly higher helical content and is less bent, indicating that strain was present in the micelle-bound helix. In the vicinity of the A30P-induced perturbations, the underlying micelle environment has rearranged, but nevertheless all aS variants maintain similar interrelationships with the micelle. Moreover, aS-micelle immersion correlates well with fast and slow aS backbone dynamics, allowing a rare insight into protein-micelle interplay.Parkinson disease (PD) 3 is characterized by the selective demise of neurons of the substantia nigra pars compacta, leading to progressive motoric dysfunction (1-3). Cell death occurs as a result of the accumulation of intraneuronal inclusions known as Lewy bodies (ubiquinated protein deposits in the cytoplasm) and Lewy neurites (thread-like proteinaceous inclusions within neurites). Biochemical and histological analyses have identified the 140-residue protein ␣-synuclein (aS) to be a major component of Lewy bodies and Lewy neurites (4, 5). Furthermore, aS gene triplication as well as any of the mutations A30P, E46K, and A53T have been genetically linked to familial PD (6 -9). At the molecular level, misfolding of aS into aggregates appears to be a common denominator in the pathogenesis of PD, which strongly correlates with age. Misfolding benefits from the impaired degradation of aS (10) and, in particular, oxidation of aS by reactive oxygen species (11,12) created by impairments in mitochondrial complex I activity (11), caused, for example, by environmental toxins, and catalyzed by iron and copper. Moreover, the dopamine-containing neurons of the substantia nigra pars compacta exhibit a unique sensitivity to impairments in mitochondrial complex I activity (11).In aqueous solution aS is predominantly unfolded but readily associates with small unilamellar vesicles (SUV) and micelles containing negatively charged lipids and detergents, respectively (13-16), supporting its association with presynaptic vesicles in vivo and rationalizing its localization primarily at axon termini (17, 18). In complex with SUV of 300 -400 Å diameter, the repeat region of aS ( Fig. 1) is likely to form a single, uninterrupted ␣-helix (19), whereas in complex with smaller diameter micelles the repeat region is partitioned into two anti-parallel ␣-helices, helix-N (Val 3 -Val 37 ) an...

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

“…We find that the TFE-induced folding landscapes for the A30P, A53T, and E46K mutants are nearly identical to WT αS, which is in accordance with previous research that showed that the pH-and temperature-induced secondary structural conformations are similar for A30P, A53T, and WT αS (34). All three mutants have also been observed to undergo similar structural transitions to WT αS in the presence of detergents or lipids (35)(36)(37), although the A30P mutation may lead to a slight local reduction in helical structure. Thus, secondary structural transitions appear to be largely similar among αS mutants.…”

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

“…This experimental design allowed us to test the effect of liposomes on FRET between any given pair of Alexa 488-and Alexa 546-labeled α-synucleins. In these experiments, we argued that multimerization of α-synuclein should only occur in the presence of negatively charged phospholipids because α-synuclein only binds to charged phospholipids (19,20,(27)(28)(29)(30). Thus, the effect of the neutral liposomes-if any-provides a tool to distinguish specific from nonspecific effects.…”

“…His-tagged TEV protease was removed using Ni-NTA agarose (Qiagen). For FRET experiments, 2.5 μg of Alexa 488-labeled α-synuclein and 2.5 μg of Alexa 546-labeled α-synuclein were incubated with and without 100 μg of SNAP- 25 Syb2 Syp1 1 1 3 1 1 9 7 5 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 3 1 3 3 3 5 Gradient fraction [23][24][25][26][27][28][29][30][31][32]. Equal volumes of each fraction were analyzed by immunoblotting (Syb2, synaptobrevin-2; α-Syn, α-synuclein; Synt1, syntaxin-1; Syp1, synaptophysin 1).…”

“…α-Synuclein is thought to adopt a bent, hairpin-like conformation on the lipid surface that is composed of two α-helices connected with a flexible linker (27)(28)(29)52). The pattern of positive vs. negative FRET pairs of labeled α-synuclein molecules allows mapping the interactions of α-synuclein molecules in the membrane-bound multimer (Fig.…”

“…The labile association of α-synuclein with membranes (23,24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25)(26)(27)(28)(29)(30)(31)(32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs.…”