**7. Structural properties of membrane-bound α-synuclein**

High resolution structural and dynamics information of α-synuclein in its lipid-bound state appear to be sufficient for the development of a better understanding of the physiological role of α-synuclein, as well as to identify the structural features that appear to be relevant to α-synuclein misfolding (Ulmer et al., 2005). However, despite the abundance of structural information for soluble proteins, relatively little is known about the structures of membraneassociated proteins in the physiologically important lipid bilayer environment (Jao et al., 2008). Consistent with this, the conformation of membrane-bound α-synuclein still remains unclear and somewhat contradictory.

Several biophysical methods have provided valuable insights into the structural features of the disordered and folded α-synuclein, including circular dichroism spectroscopy (Davidson et al., 1998; Chandra et al., 2003; Perrin et al., 2000), fluorescence spectroscopy (Rhoades et al., 2006), NMR (Bisaglia et al., 2005; Bussel & Eliezer, 2001, 2003; Bussel et al., 2005; Chandra et al., 2003; Dedmon et al., 2005; Eliezer et al., 2001; Ulmer et al., 2005), and EPR (Bortolus et al., 2008; Drescher et al., 2008; Georgieva et al., 2008; Jao et al., 2004, 2008). Binding of α-synuclein to anionic membranes induces folding of its N-terminal part into an amphipathic helix, whereas the C-terminus (residues ~98-140) remains unstructured (Bisaglia et al., 2005; Bussel & Eliezer, 2003; Chandra et al., 2003; Davidson et al., 1998; Eliezer et al., 2001; Ulmer et al., 2005). The helical content of α-synuclein is much lower in buffer and in the presence of zwitterionic membranes (Davidson et al., 1998; Zhu & Fink; 2003).

It has generally been proposed that the natural binding target of α-synuclein *in vivo* is the synaptic vesicles, the surface topology of which is most closely approximated *in vitro* by synthetic lipid vesicles (Bisaglia et al., 2005; Bussel & Eliezer, 2003; Bussell et al., 2005; Chandra et al., 2003; Jao et al., 2004; Ulmer et al., 2005). The slow tumbling rate of intact phospholipid vesicles has hindered direct studies of the vesicle-bound conformation of αsynuclein using solution NMR methods (Georgieva et al., 2008). Consequently, most of the structural information available concerns studies where detergent micelles were used as membrane-mimetic environments, because their small size facilitates high-resolution structural analysis by NMR. The conformation of micelle-bound α-synuclein has thus been thoroughly investigated, and there is a general consensus on the presence of two curved helices, with a break in the α-synuclein 38−44 region (Bisaglia et al., 2005; Bussell & Eliezer, 2003; Chandra et al., 2003; Ulmer et al., 2005). On the contrary, the structure of α-synuclein bound to lipid vesicles, which would be more relevant physiologically, remains a matter of debate. EPR analyses of α-synuclein derivatives bound to SUVs have provided evidence for

spontanous interaction with artificial membranes. Here, addition of a cytosolic preparation to the artificial membranes resulted in similar binding of α-synuclein as for biological

Lipid rafts contain a lot of the ganglioside GM1, and it has been suggested that the gangliosides mediate or facilitate the association of α-synuclein with neuronal membranes (Martinez et al., 2007). However, recently Di Pasquale et al. identifed a ganglioside-binding domain in α-synuclein that showed a marked preference for interactions with GM3, which is a minor brain ganglioside for which the expression increases with age; the Lys34 and Tyr39 residues were shown to have critical roles in the GM3 recognition by α-synuclein (Di

High resolution structural and dynamics information of α-synuclein in its lipid-bound state appear to be sufficient for the development of a better understanding of the physiological role of α-synuclein, as well as to identify the structural features that appear to be relevant to α-synuclein misfolding (Ulmer et al., 2005). However, despite the abundance of structural information for soluble proteins, relatively little is known about the structures of membraneassociated proteins in the physiologically important lipid bilayer environment (Jao et al., 2008). Consistent with this, the conformation of membrane-bound α-synuclein still remains

Several biophysical methods have provided valuable insights into the structural features of the disordered and folded α-synuclein, including circular dichroism spectroscopy (Davidson et al., 1998; Chandra et al., 2003; Perrin et al., 2000), fluorescence spectroscopy (Rhoades et al., 2006), NMR (Bisaglia et al., 2005; Bussel & Eliezer, 2001, 2003; Bussel et al., 2005; Chandra et al., 2003; Dedmon et al., 2005; Eliezer et al., 2001; Ulmer et al., 2005), and EPR (Bortolus et al., 2008; Drescher et al., 2008; Georgieva et al., 2008; Jao et al., 2004, 2008). Binding of α-synuclein to anionic membranes induces folding of its N-terminal part into an amphipathic helix, whereas the C-terminus (residues ~98-140) remains unstructured (Bisaglia et al., 2005; Bussel & Eliezer, 2003; Chandra et al., 2003; Davidson et al., 1998; Eliezer et al., 2001; Ulmer et al., 2005). The helical content of α-synuclein is much lower in buffer and in the presence of zwitterionic membranes (Davidson et al., 1998; Zhu & Fink;

It has generally been proposed that the natural binding target of α-synuclein *in vivo* is the synaptic vesicles, the surface topology of which is most closely approximated *in vitro* by synthetic lipid vesicles (Bisaglia et al., 2005; Bussel & Eliezer, 2003; Bussell et al., 2005; Chandra et al., 2003; Jao et al., 2004; Ulmer et al., 2005). The slow tumbling rate of intact phospholipid vesicles has hindered direct studies of the vesicle-bound conformation of αsynuclein using solution NMR methods (Georgieva et al., 2008). Consequently, most of the structural information available concerns studies where detergent micelles were used as membrane-mimetic environments, because their small size facilitates high-resolution structural analysis by NMR. The conformation of micelle-bound α-synuclein has thus been thoroughly investigated, and there is a general consensus on the presence of two curved helices, with a break in the α-synuclein 38−44 region (Bisaglia et al., 2005; Bussell & Eliezer, 2003; Chandra et al., 2003; Ulmer et al., 2005). On the contrary, the structure of α-synuclein bound to lipid vesicles, which would be more relevant physiologically, remains a matter of debate. EPR analyses of α-synuclein derivatives bound to SUVs have provided evidence for

**7. Structural properties of membrane-bound α-synuclein** 

membranes (Kim et al., 2006).

unclear and somewhat contradictory.

Pasquale et al., 2010).

2003).

an elongated helical structure that is devoid of any significant tertiary packing (Jao et al., 2004), or they have suggested a broken helical structure (Bortolus et al., 2008; Drescher et al., 2008). A number of recent studies have highlighted the ongoing debate regarding the physiologically relevant form, as the bent or extended membrane-bound helix (Figure 2).

Fig. 2. Illustrations of the generally proposed α-synuclein structures on (a) micelles (bent helix model) and (b) SUVs (elongated helix model) (Jao et al., 2008; Trexler & Rhoades, 2009).

#### **7.1 Helix periodicity**

Several studies have raised the question of the periodicity of the helix that is formed upon binding of α-synuclein to membranes. As indicated above, the N-terminus of α-synuclein contains seven imperfect 11-residue repeats. Using site-directed spin-labeling, it has been shown that repeats 5–7 of α-synuclein are bound to SUVs with an 11/3 periodicity (11 residues to complete three full turns) (Jao et al., 2004)*.* Sodium dodecyl sulphate (SDS) micelle-bound α-synuclein shows the same periodicity, as opposed to the 18/5 periodicity of an ideal α-helix (Bussell et al., 2005). In this ideal 18/5 periodicity, there are 3.6 residues per turn and the rotation per residue is 100°. In the α-synuclein 11/3 periodicity, the number of residues per turn is 3.67 and the rotation per residue is 98.18°. Using theoretical methods, it has indeed been concluded that the periodicity of α-synuclein is 11/3, and that through the positioning of the charged residues, this has implications for α-synuclein membrane binding. These calculations show that the energy penalty for a change in periodicity from the 18/5 to 11/3 on anionic membranes is overcome by the favorable solvation energy (Mihajlovic & Lazaridis, 2008).

#### **7.2 Analysis of α-synuclein structure by nuclear magnetic resonance**

Eliezer et al. were the first to use NMR spectroscopy to characterize the conformational properties of α-synuclein when bound to lipid vesicles and lipid-mimetic detergent micelles. They demonstrated that only the first 100 residues of the N-terminal region of α-synuclein bind to both SDS micelles and PA/PC vesicles and fold into an amphipathic helix, while the acidic C-terminal region of α-synuclein remains unstructured (Eliezer et al., 2001).

Ulmer et al. have described the structure and dynamics of α-synuclein in the micelle-bound form according to solution NMR spectroscopy. In binding to SDS micelles or SDS micelles with dodecylPC (DPC), α-synuclein forms two curved α-helices (Figure 3), helix-N (Val3-Val37) and helix-C (Lys45-Thr92). These helices are connected by a well-ordered, extended linker in an unexpected anti-parallel arrangement, which is followed by another short extended region (Gly93-Lys97) that overlaps with a chaperone-mediated autophagy recognition motif and a predominantly unstructured highly mobile tail (Asp98-Ala140) (Ulmer et al., 2005).

Alpha-Synuclein Interactions with Membranes 101

EPR analysis of 47 singly labeled α-synuclein mutants has shown that the membrane interactions are mediated by major conformational changes within seven of the N-terminal 11-amino-acid repeats: these reorganize from highly dynamic structures into an elongated helical structure. The equivalent positions within each of these different repeats are located in structurally comparable positions with respect to the membrane proximity, which suggests a curved membrane-dependent α-helical structure of α-synuclein, wherein each of these 11-aminoacid repeats takes up three helical turns (Jao et al., 2004). The α-synuclein helix is over 90 amino acids in length and it extends parallel to the curved membrane in a manner that allows the conserved Lys and Glu residues to interact with the zwitterionic headgroups, while the uncharged residues penetrate into the acyl-chain region (Jao et al., 2008). This structural arrangement is significantly different from that of α-synuclein in the presence of the commonly used membrane-mimetic detergent, SDS (Bisaglia et al., 2005, Ulmer et al., 2005). Thus these structural analyses also show that it is important to consider the lipid composition of any given bilayer, as this can have pronounced effects on the

Several other independent studies have appeared, with contradictory results. In one such study (Bortolus et al., 2008), the 35-43 region of α-synuclein bound to SUVs and to SDS micelles was investigated using site-directed spin labeling and EPR spectroscopy. The distance distributions were compatible with the presence of conformational disorder in this region, rather than for the formation of a continuous helical structure. These data showed that α-synuclein shows very similar behavior in micelles and in SUVs, and they ruled out an unbroken helical structure of the region around residue 40. This propensity for helix breaking was confirmed by their molecular dynamics simulations of the 31-52 fragment

In a study by Drescher et al. (2008), four α-synuclein mutants were prepared by inserting Cys residues labeled with the spin-label reagent (*S*-(2,2,5,5-tetramethyl-2,5-dihydro-1Hpyrrol-3-yl)methyl methanesulfonothioate) (MTSL), with each containing one label in the proposed helix 1, and a second label in helix 2. Between the labeled Cys residues within the molecule the distance resulting from their binding to the membrane was measured using dual-frequency pulsing EPR (double electron-electron resonance). Consistent with a previous report (Bortolus et al., 2008), these data showed that α-synuclein even adopts a two-helix, antiparallel arrangement on vesicles that are large enough to accommodate an extended helix, which suggests that this bent structure is also the preferred conformation of

Also using pulsed dipolar EPR, Georgieva et al. came to somewhat different conclusions. Here the distances measured between the pairs of nitroxide spin labels introduced were close to those expected for a single continuous helix. To circumvent problems associated with SUVs and rodlike SDS micelles, here they used lipid bicelles (providing a lipid-bilayer structure, yet having a particle size nearly as small as that of micelles), which produced very similar results to liposomes while offering a major improvement in experimentally accessible distance ranges and resolution. According to these data, they suggested that when α-synuclein is bound to SUVs, it forms a single α-helix, without the intermediate region of the interruption. The idea that α-synuclein can interconvert between these broken and extended helical forms was also suggested, and it thus remains possible that *in vivo* αsynuclein occupies one or the other form depending on conditions (Georgieva et al., 2008).

**7.3 Analysis of α-synuclein structure by electron paramagnetic resonance** 

protein and bilayer structures (Jao et al., 2008).

interacting with a lipid bilayer (Bortolus et al., 2008).

α-synuclein on larger vesicles (Drescher et al., 2008).

Fig. 3. Structure of α-synuclein bound to SDS micelles. Picture represents the two curved αhelices (Val3-Val37 and Lys45-Thr92), connected by extended linker. The disordered Cterminus has been partially omitted. The image was generated from the PDB (accession number 1XQ8).

Although the presence of the helix break in the micelle-bound state of α-synuclein has been suggested to be a consequence of the small size of the micelle (Jao et al., 2004), the wellordered conformation of the helix-helix connector indicates a defined interaction of αsynuclein with the lipid surfaces, suggesting that when it is bound to larger diameter synaptic vesicles, this can act as a switch between this broken helix structure and the uninterrupted helix structure (Ulmer et al., 2005). Therefore, the presence or absence of the helical break in α-synuclein appears to be the more controversial structural feature of αsynuclein when bound to lipids (Bisaglia et al., 2009). Other studies have also shown that there are two helical regions in the N-terminal sequence of α-synuclein that are interrupted by a single helix break around residue 42 (Bisaglia et al., 2005; Bussel & Eliezer, 2003; Chandra et. al., 2003). Data from Bisaglia et al. show that the region of residues 61-95 (the NAC region) is partially embedded in the micelle (Bisaglia et al., 2005)

Analysis of the dynamic processes of the α-synuclein backbone on a fast timescale (picoseconds to nanoseconds) revealed the presence of three distinct helical regions that have greater mobility with respect to the other helical fragments: Ala30-Val37, Asn65-Val70, and Glu83-Ala89 (Ulmer et al., 2005). All three of these regions have two Gly residues in close sequential proximity, which might serve to mitigate a possible effect of α-synuclein binding on membrane fluidity. The helix curvature is significantly less than predicted based on the native globular micelle shape, which indicates a deformation of the micelle by α-synuclein. Ulmer et al. suggested that the interactions of the positively charged Lys side chains, which emanate sidewards from the helices, with the negatively charged headgroups of SDS can lead to the deformation of the globular micelle along the helix axes, to form a prolate, ellipsoid particle (Ulmer et al., 2005).

As indicated above, there are four Tyr residues in α-synuclein. One of these, Tyr39, is located in the break region. Bisaglia et al. (2005) suggested that this Tyr39 is buried in the SDS micelle and proposed that this insertion might protect α-synuclein from aggregation (Zhou & Freed, 2004; Ulrih et al., 2008), as well as to protect Tyr39 from phosphorylation by p72syk tyrosine kinase (Negro et al., 2002, as cited in Bisaglia et al., 2005). This is in contrast with other models that have predicted that this Tyr39 is located either on the hydrophilic side of the helix or at the membrane-water interface (Bussell & Eliezer, 2003; Chandra et al., 2003; Jao et al., 2004; Mihajlovic & Lazaridis, 2008).

Fig. 3. Structure of α-synuclein bound to SDS micelles. Picture represents the two curved αhelices (Val3-Val37 and Lys45-Thr92), connected by extended linker. The disordered Cterminus has been partially omitted. The image was generated from the PDB (accession

Although the presence of the helix break in the micelle-bound state of α-synuclein has been suggested to be a consequence of the small size of the micelle (Jao et al., 2004), the wellordered conformation of the helix-helix connector indicates a defined interaction of αsynuclein with the lipid surfaces, suggesting that when it is bound to larger diameter synaptic vesicles, this can act as a switch between this broken helix structure and the uninterrupted helix structure (Ulmer et al., 2005). Therefore, the presence or absence of the helical break in α-synuclein appears to be the more controversial structural feature of αsynuclein when bound to lipids (Bisaglia et al., 2009). Other studies have also shown that there are two helical regions in the N-terminal sequence of α-synuclein that are interrupted by a single helix break around residue 42 (Bisaglia et al., 2005; Bussel & Eliezer, 2003; Chandra et. al., 2003). Data from Bisaglia et al. show that the region of residues 61-95 (the

Analysis of the dynamic processes of the α-synuclein backbone on a fast timescale (picoseconds to nanoseconds) revealed the presence of three distinct helical regions that have greater mobility with respect to the other helical fragments: Ala30-Val37, Asn65-Val70, and Glu83-Ala89 (Ulmer et al., 2005). All three of these regions have two Gly residues in close sequential proximity, which might serve to mitigate a possible effect of α-synuclein binding on membrane fluidity. The helix curvature is significantly less than predicted based on the native globular micelle shape, which indicates a deformation of the micelle by α-synuclein. Ulmer et al. suggested that the interactions of the positively charged Lys side chains, which emanate sidewards from the helices, with the negatively charged headgroups of SDS can lead to the deformation of the globular micelle along the helix axes, to form a prolate,

As indicated above, there are four Tyr residues in α-synuclein. One of these, Tyr39, is located in the break region. Bisaglia et al. (2005) suggested that this Tyr39 is buried in the SDS micelle and proposed that this insertion might protect α-synuclein from aggregation (Zhou & Freed, 2004; Ulrih et al., 2008), as well as to protect Tyr39 from phosphorylation by p72syk tyrosine kinase (Negro et al., 2002, as cited in Bisaglia et al., 2005). This is in contrast with other models that have predicted that this Tyr39 is located either on the hydrophilic side of the helix or at the membrane-water interface (Bussell & Eliezer, 2003; Chandra et al., 2003;

NAC region) is partially embedded in the micelle (Bisaglia et al., 2005)

ellipsoid particle (Ulmer et al., 2005).

Jao et al., 2004; Mihajlovic & Lazaridis, 2008).

number 1XQ8).

#### **7.3 Analysis of α-synuclein structure by electron paramagnetic resonance**

EPR analysis of 47 singly labeled α-synuclein mutants has shown that the membrane interactions are mediated by major conformational changes within seven of the N-terminal 11-amino-acid repeats: these reorganize from highly dynamic structures into an elongated helical structure. The equivalent positions within each of these different repeats are located in structurally comparable positions with respect to the membrane proximity, which suggests a curved membrane-dependent α-helical structure of α-synuclein, wherein each of these 11-aminoacid repeats takes up three helical turns (Jao et al., 2004). The α-synuclein helix is over 90 amino acids in length and it extends parallel to the curved membrane in a manner that allows the conserved Lys and Glu residues to interact with the zwitterionic headgroups, while the uncharged residues penetrate into the acyl-chain region (Jao et al., 2008). This structural arrangement is significantly different from that of α-synuclein in the presence of the commonly used membrane-mimetic detergent, SDS (Bisaglia et al., 2005, Ulmer et al., 2005). Thus these structural analyses also show that it is important to consider the lipid composition of any given bilayer, as this can have pronounced effects on the protein and bilayer structures (Jao et al., 2008).

Several other independent studies have appeared, with contradictory results. In one such study (Bortolus et al., 2008), the 35-43 region of α-synuclein bound to SUVs and to SDS micelles was investigated using site-directed spin labeling and EPR spectroscopy. The distance distributions were compatible with the presence of conformational disorder in this region, rather than for the formation of a continuous helical structure. These data showed that α-synuclein shows very similar behavior in micelles and in SUVs, and they ruled out an unbroken helical structure of the region around residue 40. This propensity for helix breaking was confirmed by their molecular dynamics simulations of the 31-52 fragment interacting with a lipid bilayer (Bortolus et al., 2008).

In a study by Drescher et al. (2008), four α-synuclein mutants were prepared by inserting Cys residues labeled with the spin-label reagent (*S*-(2,2,5,5-tetramethyl-2,5-dihydro-1Hpyrrol-3-yl)methyl methanesulfonothioate) (MTSL), with each containing one label in the proposed helix 1, and a second label in helix 2. Between the labeled Cys residues within the molecule the distance resulting from their binding to the membrane was measured using dual-frequency pulsing EPR (double electron-electron resonance). Consistent with a previous report (Bortolus et al., 2008), these data showed that α-synuclein even adopts a two-helix, antiparallel arrangement on vesicles that are large enough to accommodate an extended helix, which suggests that this bent structure is also the preferred conformation of α-synuclein on larger vesicles (Drescher et al., 2008).

Also using pulsed dipolar EPR, Georgieva et al. came to somewhat different conclusions. Here the distances measured between the pairs of nitroxide spin labels introduced were close to those expected for a single continuous helix. To circumvent problems associated with SUVs and rodlike SDS micelles, here they used lipid bicelles (providing a lipid-bilayer structure, yet having a particle size nearly as small as that of micelles), which produced very similar results to liposomes while offering a major improvement in experimentally accessible distance ranges and resolution. According to these data, they suggested that when α-synuclein is bound to SUVs, it forms a single α-helix, without the intermediate region of the interruption. The idea that α-synuclein can interconvert between these broken and extended helical forms was also suggested, and it thus remains possible that *in vivo* αsynuclein occupies one or the other form depending on conditions (Georgieva et al., 2008).

Alpha-Synuclein Interactions with Membranes 103

death, in amyloid diseases. Protofibrillar or fibrillar α-synuclein results in a much more rapid destruction of membranes than soluble monomeric α-synuclein, which indicates that protofibrils or fibrils are likely to be significantly neurotoxic. Further studies of α-synuclein interactions with membranes are still very important to provide us with a fuller undertanding of the molecular mechanisms of its implications in Parkinson's disease.

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