Dynamics of a-synuclein aggregation and inhibition of pore-like oligomer development by b-synuclein Igor F. Tsigelny 1,2 , Pazit Bar-On 3 , Yuriy Sharikov 2 , Leslie Crews 4 , Makoto Hashimoto 3 , Mark A. Miller 2 , Steve H. Keller 5 , Oleksandr Platoshyn 5 , Jason X J. Yuan 5 and Eliezer Masliah 3,4 1 Departments of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA 2 San Diego Super Computer Center, University of California San Diego, La Jolla, CA, USA 3 Department of Neurosciences, University of California San Diego, La Jolla, CA, USA 4 Department of Pathology, University of California San Diego, La Jolla, CA, USA 5 Department of Medicine, University of California San Diego, La Jolla, CA, USA In recent years, new hope for understanding the patho- genesis of Parkinson’s disease (PD) and Lewy body dementia (LBD) has emerged with the discovery of mutations and duplications in the a-synuclein (a-syn) gene that are associated with rare familial forms of Parkinsonism [1–3]. Moreover, it has been shown that a-syn is centrally involved in the pathogenesis of both sporadic and inherited forms of PD and LBD because this molecule accumulates in Lewy bodies (LBs) [4–6], synapses, and axons, and its expression in transgenic (tg) mice [7–9] and Drosophila [10] mimics several aspects of PD. The mechanisms through which a-syn leads to neu- rodegeneration and the characteristic symptoms of LBD are unclear. However, recent evidence indicates that abnormal accumulation of misfolded a-syn in the Keywords cation channels; modeling; molecular dynamics; oligomers; synuclein Correspondence E. Masliah, Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093–0624, USA Fax: +1 858 5346232 Tel: +1 858 5348992 E-mail: emasliah@UCSD.edu (Received 1 December 2006, revised 26 January 2007, accepted 8 February 2007) doi:10.1111/j.1742-4658.2007.05733.x Accumulation of a-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson’s disease and Lewy body demen- tia. In contrast, b-synuclein (b-syn), a close homologue, does not aggregate and reduces a-synuclein (a-syn)-related pathology. Although considerable information is available about the conformation of a-syn at the initial and end stages of fibrillation, less is known about the dynamic process of a-syn conversion to oligomers and how interactions with antiaggregation chaper- ones such as b-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of a-syn showed that a-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating a-syn dimers on the membrane incorporate additional a-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In con- trast, b-syn dimers do not propagate and block the aggregation of a-syn into ring-like oligomers. Under in vitro cell-free conditions, a-syn aggre- gates formed ring-like structures that were disrupted by b-syn. Similarly, cells expressing a-syn displayed increased ion current activity consistent with the formation of Zn 2+ -sensitive nonselective cation channels. These results support the contention that in Parkinson’s disease and Lewy body dementia, a-syn oligomers on the membrane might form pore-like struc- tures, and that the beneficial effects of b-synuclein might be related to its ability to block the formation of pore-like structures. Abbreviations aa, amino acid; a-syn, a-synuclein; b-syn, b-synuclein; GFP, green fluorescent protein; LBD, Lewy body disease; PD, Parkinson’s disease; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; tg, transgenic. 1862 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS synaptic terminals and axons plays an important role [11–14]. These studies suggest that a-syn oligomers and protofibrils rather than fibrils might be the neurotoxic species [15]. a-syn is an abundant presynaptic molecule [16] that probably plays a role in modulating vesicular synaptic release [17]. Synucleins belong to a family of related proteins including a-, b-, and c-synucleins. a-syn belongs to a class of so-called ‘naturally unfolded pro- teins’ [13,18]. Such proteins do not have a stable ter- tiary structure and during their existence change their conformations. Human a-syn is a 140-amino acid (aa) protein, and b-syn is a 134-aa protein. Each of the synucleins is composed of an N-terminal lipid-binding domain containing 11 residue repeats and a C-terminal acidic domain that has been proposed to be involved in protein–protein interactions. It has been shown [19– 22] that at the lipid–protein interface, a-syn has a conformation characterized by two helical domains interrupted by a short nonhelical turn. a-syn contains a highly amyloidogenic hydrophobic domain in the N-terminus region (aa 60–95), which is partially absent in b-syn and might explain why b-syn has a reduced ability to self-aggregate and form oligomers and fibrils [23,24]. Interestingly, although under physiological conditions b-syn is nonamyloidogenic, a recent study demonstrated that certain factors, namely, particular metals and pesticides, can cause rapid fibrillation of this molecule and of mixtures of a- and b-syn [25] under in vitro cell-free conditions. However, previous studies have shown that in the absence of metals, b-syn interacts with a-syn and is capable of preventing a-syn aggregation and related deficits both in vitro and in vivo [23,24]. Various lines of evidence support the contention that abnormal aggregates arise from a partially folded intermediate precursor that contains hydrophobic pat- ches. It has been proposed that the intermediate a-syn oligomers form annular protofibrils and pore-like structures [26–29]. The mechanism through which monomeric a-syn converts into a toxic oligomer and later into fibrils is currently under intense investiga- tion. Recent reviews indicate that the kinetics of a-syn fibrillation are consistent with a nucleation-dependent mechanism for which a partially folded intermediate is needed in the early stages of aggregation [30]. Factors leading to the formation of the folded intermediates include oxidation, phosphorylation, mutations, and lipids in the membrane [30–34]. a-syn oligomerization might occur on the membrane and involves interac- tions between hydrophobic residues of the amphipathic a-helices of a-syn [35]. These studies indicate that the hydrophobic lipid binding domains in the N-terminal region might be important in modulating a-syn aggre- gation [13,36–38]. There are several studies describing the effects of membranes and membrane-like structure on aggregation [21,39,40], however, less is known about the effects of membrane lipids on b-syn struc- ture. In this context, a recent study has analyzed by NMR the micelle-bound structure and dynamics of b- and c-syn [41]. Thus, better understanding of the steps involved in the process of a-syn aggregation is important in order to develop intervention strategies that might prevent or reverse a-syn oligomerization and toxic conversion. The conformational state of a-syn at the initial and end stages of fibrillation have been characterized in some detail and recent studies have shown that early stage oligomers are globular structures with variable height (2–6 nm) that after prolonged incubation results in the formation of elongated protofibrils which disap- pear upon fibril formation [42]. However, less is known about the dynamic process of conversion of a-syn at earlier stages and how inter- actions with antiaggregation chaperones such as b-syn and heat-shock proteins might occur. This is in part due to the transient nature of the oligomers and the difficulties in crystallizing such conformers. Therefore, the use of computer-based molecular modeling tech- niques in combination with biochemical and cell based assays might facilitate understanding the dynamic characteristics and structure of the synuclein aggre- gates. In this context, the main objective was to develop a dynamical model for the early steps of a-syn aggregation using computer simulations that includes the process of membrane docking and the potential mechanisms through which b-syn blocks a-syn aggre- gation. Our studies suggest that at early stages, propaga- ting a-syn dimers immersed in the membrane lead to the formation of pentamers and hexamers with a pore-like structure. These ring-like aggregates might correspond to Zn 2+ -sensitive nonselective cation channels whose formation is blocked by b-syn. The inhibitory effect of b-syn may result from its interac- tion with a-syn, which prevents formation of func- tional a-syn channels. Results Conformational diversity of a-syn and b-syn molecules during molecular dynamics simulations To better understand the conformational changes that a- and b -syn undergo over time and to model the homo and heterodimeric interactions preventing or I. F. Tsigelny et al. Modeling of a-syn oligomer formation FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1863 leading to aggregation, molecular dynamics simula- tions in water were performed based on the micelle- bound structure of a-syn as resolved by NMR. This approach allows the investigation of the dynamic structural changes of the folded a-syn (micelle-derived) under simplified conditions. The curved N-terminal domain of this structure is divided into two regions (termed helix-N and helix-C) [21] connected by a short linker (Fig. 1A,B). In our baseline models, the two curved helical N-terminal domains of the micelle- derived a-syn molecular structure form an angle around 55 ± 3° that decreases to around 42–44° dur- ing the first 2.0 ns of the simulation, and then increa- ses to 64–70° after 3.0–5.0 ns of simulation. During simulation (Fig. 1A,B), the initial two curved helical N-terminal domains (helices N and C) of a-syn trans- form into three uncurved N-terminal helical structures. The third helical region appears when the second curved helix (aa 46–84) converts into two uncurved helices, helix 2 (aa 46–63) and helix 3 (aa 74–84), linked by aa 64–73 (Fig. 1A,B). To confirm these results, we repeated the simulation in water, with dif- ferent seed numbers, for 3.0 ns. These additional data corroborate the initial results, showing that over time a-syn acquires a more three-dimensional shape due to movement of the C-terminal domain relative to the N-terminus (Fig. 1A,B). It is worth noting that the micelle-derived helical structure of a-syn is highly sta- ble and did not return to an unfolded state even though the molecular dynamics simulations were per- formed using the water box to simplify the procedure. At time zero, b-syn has a structural organization close to the initial structure of a-syn (Fig. 1C,D). Unlike a-syn, the curved helix at residues 46–84 of b- syn does not undergo conversion into two distinct helices during the course of simulation of up to 5.0 ns. Instead, the curved helices adopt a relatively straight configuration after 2.0 ns (Fig. 1C,D) and this conformer increases in stability from 2.0 to 3.5 ns. Further simulation shows additional conform- ational changes, mostly in the C-terminal tail and the angle between the N-terminal helices (Fig, 1C,D). Compared with a-syn, the C-terminal tail of b-syn displayed greater motility. Fig. 1. Molecular dynamics simulations of a- and b-syn monomers in water. (A) Snapshots of molecular dynamics conformations of a-syn. (B) Superimposed a-syn conformers (area of superimposition: aa 1–15). (C) Snapshots of molecular dynamics conformations of b-syn. (D) Superimposed b-syn conformers (area of superimposition: aa 1–15). Modeling of a-syn oligomer formation I. F. Tsigelny et al. 1864 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS Further analysis consisted of determining changes in secondary structure of a-syn and b-syn over time. After 500 ps of simulation for a-syn a coiled region appeared, interrupting the a-helix around residue 68 (Fig. S1A). Beginning at 750 ps, turns appeared in the a-helical structure around residue 47, then after 1.0 ns this region was transformed into a p-helix (Fig. S1A). The length of this p-helix increased with time, and from 3.0 ns covered the region from residues 45–55. In another part of the sequence, a second p-helix appeared from 2.0 ns that includes residues 74–83 (Fig. S1A). Changes in b-syn secondary structure over time con- sisted of transformations from a bended a-helical structure to the structure with two straight helices with further conversion to p-helical structure around residue 30 and the N-terminus region (Fig. S1B). The C-ter- minal region beyond residue 70 showed limited chan- ges in secondary structure (Fig. S1B). Overall, b-syn underwent significantly fewer changes in secondary structure than a-syn during molecular dynamics simu- lations. Interactions of a-syn propagating dimers predict the formation of pore-like structures The first studies of the interactions of a-syn were per- formed by docking the initial structures of two a-syn monomers on a flat surface without specific limita- tions. Under these conditions, some low energy com- plexes of two molecules formed a ‘head-to-tail’ position. This configuration is not favorable for further aggregation on the membrane. The appearance of such dimeric aggregates is caused mostly by electric charge profile complementarities between the N- and C-ter- mini of a-syn monomers (Fig. 2A and 3A). These a-syn homodimers can interact with additional a-syn molecules, but further simulations indicate that the resulting higher order aggregates are not likely to pro- duce continuously propagating multimers on the mem- brane. For the nonpropagating a-syn homodimers, usually only one a-syn has the membrane binding sur- face, such as for the 1.5 ns molecular dynamics con- formers (Fig. 2A and 3A). Fig. 2. Molecular modeling of nonpropagating and propagating a-syn aggregates on the membrane. (A) a-syn minimal energy nonpropagating dimers (head-to-tail). (B) a-syn conformer at 4.0 ns oriented to the membrane surface (membrane-contacting residues depicted in orange). (C–E) Propagating a-syn multimers on the membrane at 3.5 ns (C) Dimer (D) tetramer, and (E) hexamer. Multimers can be formed by dock- ing of a-syn monomers to a-syn propagating dimers, or by addition of a-syn dimers to a-syn propagating dimers, with either scenario result- ing in the same final hexamer structure. (F) Final configuration of the hexamer after 3.5 ns on the membrane (side view). (G) Modeling of multimers at various time points between 1.5 and 4.5 ns (top view). The table to the right margin indicates the inner diameters (ID) and outer diameters (OD) of the multimers created from the conformers obtained at the various molecular dynamics (MD) time points. I. F. Tsigelny et al. Modeling of a-syn oligomer formation FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1865 As previous studies have suggested that the assembly of a-syn into toxic oligomers might involve interactions with the membrane [26,43], we proceeded to simulate the docking of a-syn conformers on a flat surface repre- senting the membrane. The a-syn conformations at 250 ps increments of molecular dynamics were docked with their surfaces facing the membrane (defined as membrane-contacting by the mapas program [44]). These membrane-contacting surfaces were distributed as expected along the N-terminal helices of the a-syn conformers (Fig. 2B). To further verify the conforma- tional changes of a-syn dimers upon interactions with the membrane, we conducted docking of two a-syn 4 ps conformers onto a 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC) membrane with a grid cell of 1A ˚ , including the membrane in calculations (Fig. 3C). The electrostatic energy of interaction is around 30–50 kcalÆmol )1 for docking of two a-syn molecules. Only minimal differences (< 10% in docking energy values) were detected between molecules docked on the flat surface and molecules docked on the POPC membrane. In general, two possible initial docking configurations for a-syn molecules on the membrane were observed. In the first one, the dimer is arranged in a head-to-tail position and additional monomers cannot easily add to this complex to propagate toward higher order aggre- gates, as low-energy binding sites do not appear to exist for consecutive docking (Fig. 2A and 3A). It is possible for weakly propagating multimers to form over time up to 4.0 ns (Fig. 3D), however, the binding energies of the growing complexes (Fig. 3F) are significantly less favorable than for propagating configurations (Fig. 3E, Fig. 3. Modeling of docking of nonpropagating and propagating a-syn dimers and multimers on the membrane. Membrane-contacting N-ter- minal (n-term) regions are designated by boxes and C-terminal (c-term) regions by lines, as viewed perpendicular to the membrane surface. For docking, the second a-syn molecule (a-syn 2) docks to the first (a-syn 1), followed by docking of the third a-syn molecule (a-syn 3) to the second, etc., considering minimal docking energies from all possible docking positions. (A) Non-propagating conformation (head-to-tail) of two a-syn monomers that prevents low-energy docking of additional monomers. (B) Propagating conformation that allows low energy dock- ing of additional monomers added sequentially (in the direction of the arrow). (C) Docking of two a-syn conformers at 4.5 ps on the POPC membrane. (D) Weakly propagating a-syn multimer, composed of four head-to-tail conformers at 4.5 ns (E) Propagating a-syn multimer, com- posed of five head-to-head conformers at 4.5 ns (F) Electrostatic energies of the complexes growing from one a-syn monomer to a five- monomer complex at 4.5 ns. The propagating multimer has more favorable electrostatic energy than the weakly propagating multimer. Modeling of a-syn oligomer formation I. F. Tsigelny et al. 1866 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS Fig. S2), and these species would represent only a small fraction of the total multimers present. Therefore, for our purposes, this head-to-tail configuration of two a-syn monomers is designated a ‘nonpropagating dimer’. In all cases, the nonpropagating interactions involve regions of the N-terminus up to residue 75 of one molecule with residues located on the C-terminal region of the second molecule. In the second configur- ation, the pair of monomers is oriented ‘head-to-head’ (with tails oriented in similar directions) allowing con- secutive docking with similar low energy for the successive molecules of a-syn. We designate this confi- guration a ‘propagating dimer’ (Figs 2C and 3B,E). Docking additional a-syn monomers (at specified time points) to a single initial propagating dimer resulted in the formation of energetically favorable trimers, tetra- mers, pentamers, and hexamers on the membrane (Figs 2C–G, 3E, and Fig. S2). We used the molecular dynamics conformations ranging from 1.5 to 5.0 ns for docking, and noted that with longer molecular dynam- ics simulation times (4.0 ns and later), more residues on the C-terminal tail (residue 110 and above) became involved in intermolecular interactions (Fig. S3). Because the tail of a-syn carries the majority of this protein’s positive charge, this might help to explain why there was a significant enhancement of a-syn dimer docking energies (and accordingly the stability of the multimers) after 4.0 ns of simulation (Table 1). More- over, Fig. S2 shows that the most stable conformation of a-syn occurs after 3.8 ns of molecular dynamics simulation time. For b-syn, the most stable conforma- tions arise between 2.2 and 3.5 ns of simulation (Fig. S2). The most probable a-syn multimers were selected based on the conformers with the most favora- ble energies of intermolecular interaction between two monomers and the most stable conformers. Six distinct possible multimers were generated as the result of ‘pro- pagating docking’ (Fig. 2G). These multimers formed low energy pentamers and hexamers with different con- figurations that generated ring-like structures with a central lumen (Fig. 2G). The most stable multimers of a-syn were generated with a-syn conformers from 4.0 ns simulation and later. The theoretical pentameric and hexameric conformations of the a-syn multimers on the membrane are reminiscent of the pore-like appearance of cell-free a-syn aggregates that have been reported by atomic force microscopy (AFM) [26]. a-syn propagating dimers form pore-like structures that are embedded in the membrane To further investigate how closely the simulation- derived model resembles a-syn aggregates generated in vitro, recombinant a-syn was incubated for various time periods at 65 °C and the preparations analyzed by western blot and electron microscopy. At 15 h of incu- bation, immunoblot analysis showed the appearance of multiple bands at molecular weight levels consistent with a-syn dimers, trimers, tetramers, and pentamers (Figs 4A,B). After 20 h, higher order aggregates consis- tent with hexamers were also detected (Fig. 4A,B). Ultrastructurally, after 10 h of incubation, ill-defined globular elements were noted, and around 15 h, ring- like structures ranging in diameter between 9 and 15 nm with a central channel of 2–5 nm were found (Fig. 4C–E), while after 20 h fibrils (9–12 nm in diam- eter) became more apparent (Fig. 4F). Remarkably, the ring-like structures that formed after 15 h of incubation were of similar dimension to the a-syn pentamers and hexamers generated after 4.0 ns of molecular dynamics simulation (Fig. 4K) and further simulations showed that they became embedded in the membrane after rel- atively short (350 ps) molecular dynamics simulation of the membrane–protein complex (Fig. 5A). During extended simulation times, the a-syn pentamer embeds progressively further into the membrane, reaching 16 A ˚ in the membrane by 800 ps (Fig. 5B–E). b-syn interrupts the formation of propagating a-syn dimers We have previously shown that b-syn is capable of reducing a-syn accumulation and related deficits [23], however, the molecular characteristics for the interac- tions between these two molecules are unclear. For this purpose, we modeled b- and b-syn, and b- and a-syn heterodimeric interactions. Firstly, theoretical docking of various molecular dynamics conformers of a-syn to conformers of b-syn was performed. All of the docked a-syn–b-syn complexes displayed a significant level of negative electrostatic energy of formation (Table 2). In these simulations, b-syn was able to bind a-syn, cre- ating stable nonpropagating heterodimers, similar to nonpropagating a-syn homodimers (Fig. 6A). Strong Table 1. Intermolecular interaction energies of propagating a-syn ⁄ a-syn dimers docked on the flat membrane. MD, molecular dynamics. MD time (ns) Electrostatic energy (kcalÆmol )1 ) 1.50 )10.6 2.00 )10.6 2.50 )13.4 3.50 )15.1 4.00 )19.7 4.50 )32.9 I. F. Tsigelny et al. Modeling of a-syn oligomer formation FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1867 electrostatic interactions contributed to the formation of these a- and b-syn heterodimers. For example, com- plexes between a-syn (2.5 ns) and b-syn (2.2 ns) dis- played a minimum intermolecular electrostatic energy of )31.6 kcalÆmol )1 , while the electrostatic energy of interaction between two a-syn (2.5 ns) conformers that can aggregate into hexamers on the membrane was )13.4 kcalÆmol )1 . Thus the binding energy between a- and b-syn was significantly lower, and more favora- ble, than the energy of interaction between two a-syn molecules located on the membrane-like surfaces. The net charge for b-syn ()16 e) at pH 7.0 is much lower than that of a-syn (– 9 e), which might help to explain why it is less likely for b-syn than for a-syn to form propagating dimers in the membrane [23,24]. In addition to binding to a-syn monomers, the simu- lations showed that b-syn interacts with a-syn mono- mers and propagating dimers (Fig. 6C), which can theoretically form annular-like structures on the mem- brane. In fact, b-syn binding to earlier a-syn conform- ers was stronger than that of the next a-syn molecule that participates in propagating membrane-facing pair- wise docking. One b-syn molecule (shown in green in Fig. 6C) docked to an a-syn dimer on the membrane has a position that conflicts with the neighboring a-syn molecules that can flank it from both sides in the poss- ible multimeric complex. Further analysis of the elec- trostatic energies of interaction between heterodimers starting with the 1.5 ns molecular dynamics conformer showed that in most cases, the energy of interaction between b-syn and a-syn (Table 2) was significantly lower than for a-syn homodimers ()10.6 kcalÆmol )1 , Table 1). This supports the possibility that b-syn might be able to interrupt the assembly of propagating a-syn homodimers at various stages of the oligomerization process. b-syn blocks the formation of a-syn ring-like structures and attenuates ion conductance alterations Previous studies have shown that when b- and a-syn are incubated simultaneously, b-syn reduces a-syn aggregation over time [23,24,45]. However, it is unclear Fig. 4. Biochemical and ultrastructural analysis of a-syn aggregation, interactions with b-syn, and modeling of ring-like structures. (A) In vitro cell-free aggregation of a-syn monomers into dimers, trimers, tetramers, pentamers, and hexamers over time without (left panel) and with (right panel) the addition of b-syn. (B) Semiquantitative analysis of levels of a-syn multimers over time. (C–F) Electron microscopy analysis of a-syn aggregation over time into ring-like structures and fibrils. (G–J) Electron microscopy analysis demonstrating reduction in a-syn aggrega- tion over time in the presence of b-syn. Scale bar ¼ 20 nm. (K) Superimposition of a-syn pentamer (4.5 ns) onto the ring-like structure detec- ted by electron microscopy. Scale bar ¼ 10 nm. Modeling of a-syn oligomer formation I. F. Tsigelny et al. 1868 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS whether b-syn might decrease a-syn aggregation when added after the process of a-syn oligomerization has started. The theoretical model presented in the previ- ous section predicts that under experimental in vitro conditions, addition of b-syn might prevent further aggregation of a-syn (Fig. 4). To investigate this possi- bility, a-syn was allowed to aggregate and then b-syn was added for various lengths of time. When b-syn was added 1 h after a-syn aggregation started, there was a significant decrease in the subsequent formation of a-syn multimers at the various time points analyzed (Fig. 4A,B). Consistent with the immunoblot studies, ultrastructural analysis showed that b-syn reduced the formation of globular, ring-like, and fibrillar structures (Fig. 4G–J). As previous studies have suggested that the a-syn ring-like structures might form pores in the membrane that might be responsible for the neurotoxic effects of a-syn oligomers [26,29,46,47], we investigated whether abnormally high levels of ion currents are detected in cells overexpressing a-syn and if this process might be attenuated by b-syn. For this purpose, we recorded and compared whole-cell currents in HEK293T cells transiently transduced with lentiviral vectors expressing a-syn, b-syn, or a-syn and b-syn together (Fig. 7). Immunoblot analysis confirmed that cells expressed comparable levels of a-syn and b-syn (Fig. 7A). Dou- ble-labeling verified that in cotransduced cells, green fluorescent protein (GFP) was also expressed with either a-syn or b-syn (Fig. 7B). The target cells (dis- playing green fluorescence) for electrophysiological measurements were identified by cotransduction with a lenti-GFP vector (Fig. 7C). Cells expressing a-syn Fig. 5. Modeling of the embedded a-syn complex in the membrane over time. (A) Top view (at the level of the uppermost membrane-associated atom) of the embed- ded portion of the a-syn pentamer (350 ps) on the POPC membrane (white, a -syn pen- tamer; green, membrane phospholipids). Note the penetration of the pentamer into the membrane and the exposed membrane in the center of the a-syn ring-like structure. (B–E) The steps of penetration of the a-syn pentamer into the POPC membrane during 0.8 ns molecular dynamics simulation (B, ini- tial; C, 0.2 ns; D, 0.5 ns; E, 0.8 ns). The depth of protein insertion into the mem- brane was measured between the upper- most membrane-associated atom and the atom that is embedded deepest into the membrane. Table 2. Intermolecular interaction energies of the b-syn conform- ers with 1.5 ns molecular dynamics a-syn conformer docked on the flat membrane. MD, molecular dynamics. b-syn conformer MD time (ns) Electrostatic energy (kcalÆmol )1 ) Initial )27.4 0.25 )22.0 0.50 )29.8 0.75 )45.9 1.00 )37.8 1.50 )26.7 I. F. Tsigelny et al. Modeling of a-syn oligomer formation FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1869 showed a significant increase in whole-cell currents eli- cited by depolarizing the cells from a holding potential of )50 mV to a series of test potentials ranging from )80 to +80 mV (Fig. 7D,E). The current density at +80 mV was 54.8 ± 4.3 in cells transduced with an empty vector, 181.1 ± 18.1 (P<0.001 vs. vector con- trol) picoamperes ⁄ picofarads in a-syn-expressing cells, 64.2 ± 5.3 (P ¼ 0.21) picoamperes ⁄ picofarads in cells expressing b-syn, and 78.1 ± 10.4 (P ¼ 0.07) pA ⁄ pF in cells transduced with a-syn + b-syn (Fig. 7D,E). Furthermore, the currents in a-syn transduced cells were sensitive to Zn 2+ (Fig. 7F). Extracellular applica- tion of 5 lm Zn 2+ reversibly decreased the currents; the maximal inhibition took place within 3 min (Fig. 7F). These data indicate that a-syn forms a Zn 2+ - sensitive nonselective cation channel and that coexpres- sion of a-syn with b-syn significantly inhibited the amplitude of currents of the putative a-syn channels. Discussion The present study showed by utilizing molecular mode- ling and molecular dynamics simulations, in combina- tion with biochemical and ultrastructural analysis, that a-syn can arrange into homodimers that can adopt nonpropagating and propagating conformations. The evidence predicts that propagating a-syn dimers dock on the membrane surface and can incorporate addi- tional a-syn molecules, leading to the formation of pore-like structures. In contrast, b-syn dimers do not propagate, and when interacting with a-syn aggregates block the propagation of a-syn into multimeric struc- tures. Recent studies have suggested that the transfor- mation of a-syn into a neurotoxic molecule might involve the sequential conversion of a-syn monomers into globular oligomers and then protofibrils [46]. In contrast, a-syn fibrils, which are present in the LBs [6], might represent a mechanism for isolating toxic oligo- mers [15]. Previous studies have investigated the con- formation of a-syn either at the very initial stages of aggregation [21] or during the process of fibril forma- tion [42]. In micelles, a-syn monomers consist of two curved a-helices connected by a short linker in an anti- parallel arrangement, followed by a short extended region and a predominantly unstructured mobile tail [21,48]. The molecular dynamics studies described here showed that this structure of a-syn displayed significant changes in the organization of the N-ter- Fig. 6. Molecular modeling of the interactions of b-syn with a-syn monomers and dimers. (A) a-syn and b-syn minimal energy nonpropagat- ing heterodimers. (B) Primary electrostatic interactions in the minimal energy a-syn and b-syn dimer. (C) b-syn minimal energy complex with the a-syn dimer (4.5 ns simulation for a-syn and 2.2 ns simulation for b-syn). This complex does not support further propagation. Modeling of a-syn oligomer formation I. F. Tsigelny et al. 1870 FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS minal helices from 2 to 3 helices over time, which might lead to more complex membrane interactions. Computerized analysis predicted that these changes were accompanied over time by alterations in the secon- dary structure showing that a p-helical conformation appears in the N-terminus in addition to the a-helix. However, confirmation of this structural transforma- tion awaits NMR analysis. Interestingly, molecular modeling of the misfolding of the Alzheimer’s disease amyloid-b protein has shown a rapid transition of the N-terminal a-helix 1 into a p-helix [49]. Such con- formational changes, in combination with b-hairpin structures, might be essential to the aggregation process [50] and the subsequent formation of pore-like structures. Under basal conditions, both nonpropagating and propagating dimers might exist, with a higher propor- tion of dimers exhibiting a nonpropagating conforma- tion. In disorders with a-syn aggregation such as PD it is possible that an increased proportion of propagating dimers might be present. The conditions that might favor an increased ratio of propagating a-syn com- plexes are unclear, but given the conformational insta- bility of the proteins implied by both experimental and modeling results, it may be highly sensitive to local environmental influences. In support of this, closer association with the membrane has been suggested to induce a-syn oligomerization [35]. It has been reported that small oligomeric forms of a-syn preferentially asso- ciate with lipids and cell membranes [35], however, the Fig. 7. Studies of ion conductance in a -syn and b-syn transduced cells utilizing lentiviral vectors. (A) HEK293T cells transduced with lentiviral vectors encoding a-syn, b-syn, and GFP express comparable protein levels. (B) Double-labeling immunocytochemical analysis of 293T cells cotransduced with lenti-GFP and lenti-asyn or lenti-bsyn. (C) 293T cells transduced with an empty GFP vector, lenti-asyn, or lenti-bsyn and cells cotransfected with a-syn and b-syn. The transduced cells are indicated by green fluorescence. Scale bar ¼ 20 lm. (D, E) Representa- tive currents elicited by depolarizing the cells from a holding potential of )50mv to a series of test potentials ranging from )80 to +80mv, and corresponding current–voltage relationship (E; means ± SE) in tranduced cells. (F) Representative currents at +80mv (left panel) before (Cont), during (Zn 2+ ) and after (Wash) application of 500 l M Zn 2+ . Time course (right panel) of the change in current density before, during, and after extracellular application of Zn 2+ . The arrows correspond to the currents shown in the left panel (Cont, a; Zn 2+ , b; and Washout, c). I. F. Tsigelny et al. Modeling of a-syn oligomer formation FEBS Journal 274 (2007) 1862–1877 ª 2007 The Authors Journal compilation ª 2007 FEBS 1871 [...]... Theoretical docking of synucleins to the membrane Preparation and electrophysiological analysis of cells expressing a- and b-syn Interactions between two a-syn and between a-syn and b-syn monomers were studied using programs hex 4.5 [72,73] and the Docking module of insight ii (Accelrys) ˚ ˚ with a 1 A grid and cut-off distance of 20 A Taking into account previous studies [35] showing that a-syn aggregation. .. permeabilization by protofibrillar alpha-synuclein is sensitive to Parkinson’s disease-linked mutations and occurs by a pore-like mechanism Biochemistry 41, 4595–4602 30 Fink AL (2006) The aggregation and fibrillation of alpha-synuclein Acc Chem Res 39, 628–634 31 Hashimoto M, Hernandez-Ruiz S, Hsu L, Sisk A, Xia Y, Takeda A, Sundsmo M & Masliah E (1998) Human recombinant NACP ⁄ a-synuclein is aggregated and fibrillated... induced by mutant alpha-synuclein contributes to the degeneration of neural cells J Neurochem 97, 1071–1077 48 Ulmer TS & Bax A (2005) Comparison of structure and dynamics of micelle-bound human alpha-synuclein and Parkinson disease variants J Biol Chem 280, 43179–43187 49 Urbanc B, Cruz L, Yun S, Buldyrev SV, Bitan G, Teplow DB & Stanley HE (2004) In silico study of amyloid beta-protein folding and oligomerization... alpha-synuclein in its free and lipid-associated states J Mol Biol 307, 1061–1073 40 Cole NB, Murphy DD, Grider T, Rueter S, Brasaemle D & Nussbaum RL (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alpha-synuclein J Biol Chem 277, 6344–6352 41 Sung YH & Eliezer D (2006) Secondary structure and dynamics of micelle bound beta- and gamma-synuclein Protein Sci... counter ions The namd molecular dynamics program version 2.5 [65] was used with the CHARMM27 force-field parameters [66] to simulate the behavior of a-syn and b-syn molecules in water under normal conditions and the interaction of the POPC membrane with the a-syn aggregates The temperature was maintained at 310 K by means of Langevin dynamics using a collision frequency of 1 ps)1 A fully flexible cell... Takenouchi T & Masliah E (1999) Role of cytochrome c as a stimulator of a-synuclein aggregation in Lewy body disease J Biol Chem 274, 28849–28852 33 Beyer K (2006) Alpha-synuclein structure, posttranslational modification and alternative splicing as aggregation enhancers Acta Neuropathol (Berl) 112, 237–251 34 Uversky VN (2003) A protein-chameleon: conformational plasticity of alpha-synuclein, a disordered protein...Modeling of a-syn oligomer formation I F Tsigelny et al conformations of a-syn multimers have been difficult to study due to the inability to crystallize the oligomeric form of this protein Our molecular dynamics studies support the contention that oligomers of a-syn associate with membranes and suggest that propagating dimers might be thermodynamically... 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[76] and rmsd between them was calculated The most stable MD conformations (boxed regions of the graph) begin at 3.8 ns for a-syn (red) and occur between 2.2 and 3.5 ns for b-syn (blue) Fig S3 Specific intermolecular interactions between two head-to-head a-syn monomers over time Sites of Modeling of a-syn oligomer formation interaction (arrows) between amino acids in each of the monomers (a-syn 1 and . Dynamics of a-synuclein aggregation and inhibition of pore-like oligomer development by b-synuclein Igor F. Tsigelny 1,2 ,. analyzed by NMR the micelle-bound structure and dynamics of b- and c-syn [41]. Thus, better understanding of the steps involved in the process of a-syn aggregation