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J. Biol. Chem., Vol. 280, Issue 10, 9595-9603, March 11, 2005
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-Synuclein*
From the
Laboratory of Chemical Physics, NIDDK and the ¶Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, October 18, 2004 , and in revised form, December 13, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved -helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-synuclein (aS)1 has been implicated in the molecular chain of events leading to Parkinson's disease, the second most common neurodegenerative disorder in humans (1–3). Lewy bodies and Lewy neurites found in Parkinson's disease in general contain aggregates of aS (4, 5), which is prone to form aggregates in vitro (6, 7). Moreover, three missense mutations (A30P, E46K, and A53T) in the gene encoding aS cause familial Parkinson's disease as well as aS gene triplication (3, 8, 9). The amino acid sequence of aS consists of 140 residues with 7 copies of an unusual 11-residue repeat, followed by a hydrophilic tail (Fig. 1). Based on sequence analysis, it was suggested that aS interacts with lipid membranes through its repeat region (10, 11) and interactions with small unilamellar vesicles (SUVs) and micelles preferentially containing negatively charged head groups have been documented in vitro (10, 12–14). In accordance with those properties, aS localizes in a physiological environment at nerve termini in close proximity to synaptic vesicles (15, 16) and may be implicated in synaptic plasticity and neurotransmitter release (8, 9).
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-stranded conformation in the aggregated, fibrillar forms characteristic of Lewy bodies (20), aggregation of aS into dimers and multimers is promoted by lipid environments that induce an -helical conformation (14, 21, 22). Thus, high resolution structural and dynamic information of aS in its helical conformation appears pertinent to developing a better understanding of the physiological role of aS, as well as possible structural features relevant to aS misfolding. Here, the structure and dynamics of aS in the micelle-bound form, determined by solution NMR spectroscopy, are presented and related to the vesicle-bound state. As solution NMR is limited by particle size, the direct study of vesicle-bound aS is not feasible. We therefore elected to resort to smaller diameter micelles, which elicit a similar aS helical content as SUV (12).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-synuclein (aS) gene was expressed from the kanamycin restricted, T7lac promoter controlled pET-41 vector (Novagen, Inc.). A mutant, aS(S87C), was constructed from this vector following the QuikChange protocol (Stratagene, Inc.). Both genes were overexpressed in Escherichia coli BL21(DE3) cells, cultured in M9 minimal medium (6 g/liter Na2HPO4·7H2O, 3 g/liter KH2PO4, 0.5 g/liter NaC1, 1 mM MgSO4, 100 µM CaC12·2H2O, and D-glucose/NH4Cl as specified below). 1 g/liter 98% 15NH4Cl was employed to achieve 15N enrichment. 2 g/liter 99% [13C]glucose, 1 g/liter 98% 15ND4Cl, and 1 g/liter 98% D/99% 13C/98% 15N-rich growth medium supplement (Isogro, Isotec, Inc.) in D2O solution were employed to label with 2H/13C/15N. Uniformly 2H/13C/15N-enriched protein with selectively protonated Val-
1,2, Leu-
1,2, and Ile-1 methyl groups was prepared by using 2 g/liter 97% D, 99% [13C]glucose, 1 g/liter 98% 15ND4Cl in D2O solution, and, added 45 min before protein induction, 50 mg/liter 3,3-2H2-, -[13C]ketobutyrate, and 100 mg/liter 3-2H-, -[13C]ketoisovalerate (23). Protein expression was induced at A600 = 0.8 for 2 h in H2 Oor 4 h in D2O. Purification involved heat precipitation (10 min, 80 °C) of the cells in 50 mM Tris·HCl, pH 7.5, 500 mM NaCl (2 mM -mercaptoethanol for S87C). Subsequent ion-exchange chromatography on Q-Sepharose (Amersham Biosciences) yielded >98% pure protein of the correct mass as judged by SDS-PAGE and electrospray mass spectrometry.
NMR Sample Preparation—All samples were prepared in H2O to contain aS at a concentration of 0.5 mM (
280 = 5120 M-1cm-1), 75 mM SDS, 6% D2O, and 0.02% (w/v) NaN3 in a total volume of 270 µl. Nine samples were prepared. Sample A was enriched only in 15N, sample B was uniformly enriched in 2H/13C/15N, sample C with 2H/13C/15N, but selectively protonated at Val-1,2, Leu-1,2, and Ile-1 methyl groups and also employed deuterated SDS. Samples D and E used 2H/13C/15N-enriched aS in the presence of charged stretched polyacrylamide gels (see below). Samples F–H employed 2H/13C/15N-enriched aS(S87C) tagged with cysteaminyl-EDTA, complexed with Ca2+,Mn2+, and Co2+, respectively. Samples A–E were buffered by 20 mM NaH2PO4/Na2HPO4, pH 7.4, and samples F–H by 20 mM HEPES·NaOH, pH 7.4. The last sample, I, contained 2H/13C/15N-labeled aS, buffered by 20 mM Tris·HCl, pH 8.4.
The gel for sample D was polymerized from a 4.6% w/v solution of acrylamide (AA), 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), and bisacrylamide with a monomer to cross-linker ratio of 39:1 (w/w) and a molar ratio of 96:4 of AA to AMPS. To allow easy comparison between gels containing different amounts of charged monomer, AMPS is counted here as AA and the AMPS:AA ratio is specified separately. Gel for sample E was polymerized from a 5.2% w/v solution of AA, AMPS, and piperazine diacrylamide as cross-linker with a monomer to cross-linker ratio of 39:1 (w/w) and a molar ratio of 98:2 of AA to AMPS. For the reported monomer to cross-linker ratio, piperazine diacrylamide was counted as bisacrylamide to again allow for direct comparison between gels. Polymerization was initiated by the addition of ammonium persulfate and TEMED at 0.1% w/v and v/v, respectively, and allowed to proceed in a cylinder of 5.4-mm diameter for 3 h at 23 °C. Subsequently, gels were dialyzed, dried, soaked with protein solution for 2 days, and transferred to NMR tubes as described (24, 25). 2H quadrupolar splittings of 1.2 and 1.7 Hz were measured for samples D and E, respectively.
A 20 mM HEPES·NaOH, pH 8.0, 200 mM NaCl, 25 mM EDTA, 0.02% NaN3 solution of 50 µM aS(S87C) containing 1 mM dithiothreitol was exchanged into the dithiothreitol-free solution containing 1 mM N-[S-(2-pyridylthio)cysteaminyl]-EDTA (Toronto Research Chemicals, Inc.) by ultrafiltration and allowed to react overnight at room temperature. Small amounts of homodimeric aS(S87C) were removed by ion-exchange chromatography, and aS(S87C)-cysteaminyl-EDTA was exchanged into 20 mM HEPES·NaOH, pH 7.4, 50 mM NaCl by ultrafiltration. The EDTA tag was then charged by adding a 1.2 times excess of the desired ion over the protein, washed extensively with 20 mM HEPES·NaOH, pH 7.4, 700 mM NaCl to remove non-specifically bound ions, and exchanged into 20 mM HEPES·NaOH, pH 7.4, 0.02% NaN3 by ultrafiltration. Samples were completed by the addition of SDS and D2O to 75 mM and 6%, respectively.
NMR Spectroscopy—All experiments were carried out at 25 °C on Bruker spectrometers operating at 1H frequencies of 600, 750, and 800 MHz equipped with cryogenic (600 MHz) or room temperature (750 and 800 MHz) probes. Data were processed and analyzed with the nmrPipe package (26). Throughout most experiments, the TROSY N-H component was selected (27, 28). HN, N, C, and C' assignments were made from HNCA, HN(CO)CA, HNCO, and HN(CA)CO experiments using sample B and the reported HN, N, and C assignments (12). Subsequently, H and partial H assignments were obtained from TOCSY-TROSY experiments (sample A, 
mix = 36 ms). CH3 assignments of Val-1,2, Leu-1,2, and Ile-1 moieties of sample C were based on (H)CCONH and H(C)CONH experiments (29).
HN-HN NOEs were measured from HSQC-NOESY-TROSY experiments (sample B, mix = 170 ms), HN-H NOEs from NOESY-TROSY experiments (sample A, mix = 100 ms), HN-H, (Val/Leu/Ile) NOEs from NOESY-TROSY experiments (sample C, mix = 150 ms), and CH3-CH3 NOEs from CT-HSQC-NOESY-CT-HSQC experiments (sample C, mix = 150 ms).
3JC'C and 3JNC couplings for aromatic and aliphatic residues were obtained from quantitative J-correlation spectroscopy (30, 31) with dephasing times of 60 and 160 ms, respectively. 1JNH, 1JCC', 1JC'N, and 1JNH + 1DNH, 1JCC' + 1DCC', 1JC'N + 1DC'N couplings were determined from mixed-constant time, HN-coupled HNCO, C-coupled HNCO, or quantitative J-correlation HN(CA)CO (32) and quantitative J-correlation HNCO experiments (33) of samples B, D, and E, respectively.
The 15N relaxation parameters R1, R2, and {1H}-15N NOE were determined at 60.8-MHz for sample B (34, 35). For the {1H}-15N NOE measurement, 5 s of presaturation preceded by a recycling delay of 4 s were used for the NOE experiment and a 9-s recycle delay for the reference experiment. Mn2+-induced paramagnetic relaxation enhancements (PRE) of 1HN transverse magnetizations, 
2, were determined from the difference in R2(1HN) of the Ca2+- and Mn2+-loaded samples, employing the pulse sequence described in Ref. 36. Co2+-induced pseudo-contact shifts were found to result in peak doubling of resonances close to the metal ion, indicating the presence of two diastereomers for the cysteaminyl·EDTA·Co2+ complex and, for this reason, were not pursued further.
Structure Calculation—Due to the pronounced differences in dynamics experienced by the RDC (see "Results"), the local geometry of Val3-Lys97, exhibiting general order parameters (S2) above 0.5, was generated by molecular fragment replacement (37). The remaining residues are represented by random-coil conformations. A fragment length of 7 residues, 372 H, N, C, and C' chemical shifts, and 520 1DNH, 1DCC', and 1DC'N residual dipolar couplings (RDCs) were used for molecular fragment replacement, allowing the alignment tensor for each fragment to be an adjustable parameter. For each fragment ten candidates in best agreement with the chemical shifts and RDCs were selected from the molecular fragment replacement data base (37). Backbone dihedral angles were then determined by averaging over all selected candidates of all (overlapping) fragments containing any given angle, but excluding fragments where this angle is located in its N- or C-terminal residue. The alignment tensor magnitudes, Da, calculated for this average structure varies little with fragment lengths ranging from 7 to 11 residues (Supplementary Fig. 1), indicating that dynamics is not significantly impacting the fragment structures over this length range. Standard deviations for the angles were typically between 2 and 10° for most residues, and between 10 and 25° for Ala30-Thr44. The local geometry thus defined was implemented as backbone dihedral restraints in subsequent structure calculations with the program Xplor-NIH 2.9.5 (38).
The backbone HN-HN, NOEs were found to be in good agreement with this generated structure, and side-chain conformations were restricted by HN-H(Ile/Thr), HN-H(Val/Leu) and CH3-CH3 NOEs. For residues with non-rotamer-averaged 3JC'C and 3JNC couplings, the 
1 rotamer state was selected based on those couplings (30, 31). For the translation of PRE 2 values into distances, the cysteaminyl-EDTA-Mn2+ group was represented by an ensemble of three structures during simulated annealing, and the model-free Solomon-Bloembergen equations were applied (39). The 2 values of repeat region residues exhibited a constant, reproducible background (Supplementary Fig. 2), which was subtracted (6 Hz) to achieve agreement between the position of the paramagnetic tag and helix-C, i.e. internal referencing was performed. For helix-N the HN intensity ratios between dia- and paramagnetic samples, IMn2+/ICa2+, followed the 2 values except for the first nine residues (Supplementary Fig. 2), suggesting some variation in distance between helix-N and -C with more distant conformers contributing overproportionally to the ensemble-averaged R2(Mn2+) rate, i.e. the calculated interhelical distances will be somewhat biased to more remote distances than the actual average. In order not to introduce opposing restraints, the structure was calculated by only considering the 2 values of Lys10-Lys21 of helix-N, besides the values for helix-C. It is also noted that the paramagnetic tag is clearly oriented to one side of helix-C (Supplementary Fig. 3). To aid in the placement of the helices relative to each other, global alignment tensors were used to describe RDC for residues with S2 > 0.8 and with fragment number exhibiting |Da| > 6 Hz (residues 9–23, 54–63, and 70–81 for sample D and residues 9–23, 57–63, and 70–81 for sample E).
Simulated annealing calculations were carried out from 500 to0Kin the presence of an empirical backbone-backbone hydrogen-bonding potential (40) and torsion angle potentials of mean force (41). In order not to distort helix-C by the relatively strong forces acting on the paramagnetic tag, the dihedral angle restraints of residues 85–88 were tightened. To allow a wider range of helix-helix orientations, the dihedral angle restraints of residues 39–43 were relaxed. The energy-minimized average structure, calculated from the ensemble of 20 lowest energy structures, was deposited in the protein data base under accession code 1XQ8 [PDB] .
SDS Titration—Titrations were performed in 20 mM NaH2PO4/Na2HPO4 (pH 7.4)/6% D2O at 25 °C and 800 MHz. The aS starting concentration was 0.2 mM, and 11 titrations steps were performed from aS:SDS molar ratios of 0:422 (Fig. 2). At each titration point a TROSYHSQC spectrum was recorded (t15N,max = 144.9 ms, t1HN,max = 113.9 ms). In the absence of SDS selected aS assignments were made based on an HNCA experiment and comparison of aS wild-type spectra with spectra of aS mutants A30P and A53T. Between aS:SDS ratios of 14 to 43 the assignment of a few residues with characteristic chemical shift was made. For aS:SDS ratios ranging from 58 to 422 peaks shift relatively little and assignments followed directly from the assignments of sample B (cf. above).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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70 SDS molecules (12). Consequently, up to a molar aS:SDS ratio of 1:70, multiple aS molecules may bind to a single micelle. A titration with SDS was carried out to cover molar aS:SDS ratios ranging from 0 to 422 at an initial aS concentration of 0.2 mM. Chemical shift changes of the backbone 1HN and 15N nuclei of each residue were monitored during the titration, and the 1HN chemical shift changes of four residues, Ala29, Thr54, Ala56, and Met116, are presented here (see Fig. 2). Changes in chemical shifts are sensitive indicators of changes in local structure and, thus, excellent probes of the transition of aS from the free to the micelle-bound state. The first titration step, to an aS:SDS ratio of 1:14, broadened the resonances of the entire repeat region of aS (Fig. 1) essentially beyond detection. The broadening may arise from the binding of multiple copies of aS, resulting in large particle sizes, or exchange kinetics that is intermediate on the NMR timescale. As the titration progresses (aS:SDS = 1:29), the missing resonances reappear at new positions. For residues of the C-terminal tail region (Fig. 1), no significant change in signal intensity was observed during these two titration steps, but essentially all of their very small shift changes take place there (Fig. 2D). At an aS:SDS ratio of 1:43, two species arise for each repeat region residue, one resonance that often, but not always, appears close to the previous titration step and one that is headed toward the final micelle-bound state (Fig. 2C). At aS:SDS ratios of 1:58 and higher only one resonance was observed for all residues, which asymptotically approached the final micelle-saturated state (Fig. 2, A–C). In good agreement with the previous estimate of the SDS micelle size (12), the chemical shift changes for repeat region residues leveled off rapidly after the titration point at an aS:SDS ratio of 1:73 was reached (Fig. 2, A–C), i.e. after each micelle had at most a single aS molecule bound, showing that quantitative aS·micelle complex formation had occurred.
Remarkably, for most residues of the repeat region, if not all, the chemical shift in the absence of SDS does not fall on the smooth curve toward the final micelle-saturated state (Fig. 2, A and B), indicating that the repeat region progresses through an intermediate state to reach the micelle-saturated state. Given the excess of aS over micelles, this intermediate state could involve two or more micelle-bound aS molecules. However, an analogous titration monitored by circular dichroism spectroscopy has found the aS helical content to increase until a aS:SDS ratio of 1:70 is reached (12), indicating that below this ratio the free aS state remains populated and/or that, within such higher order complexes, aS is structurally different from its final helical, micelle-bound state.
When comparing the aS backbone HN and N chemical shifts in the presence of an excess of SDS-only micelles and mixed micelles composed of DPC and SDS at a molar ratio of 70:30, most shifts agreed to within ±0.1 and ±1.0 ppm for HN and N nuclei, respectively (Fig. 3 and Supplementary Fig. 4). The magnitude of these shift changes was relatively small, suggesting that the chemical makeup of SDS micelles does not strongly affect the aS structure. The most significant deviations were observed for residues located before and after the aS repeat region (Fig. 1) as well as those near the reported interruption of helical structure around Ser42-Thr44 (12, 18). Minor structural rearrangements of these regions are a likely consequence of a change in micelle shape or diameter, or a subtle change in the interaction with the micelle itself. In conclusion, the chemical makeup of SDS micelles did not strongly affect the aS structure, and, when there was an excess of SDS micelles over aS molecules, quantitative binding occurred, allowing the structure determination of micelle-bound aS.
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, and C') and, hence, the structure of micelle-bound aS (data not shown). To introduce the Mn2+ ion at a defined site of aS, an EDTA-derived tag was introduced to S87C-substituted aS, which is otherwise devoid of Cys residues. Chemical shift changes ensuing from the S87C substitution and the presence of the tag were restricted to the immediate vicinity of residue 87 (Supplementary Fig. 5), showing that the aS structure is not unduly perturbed by the tagging.
The structure of micelle-bound aS consists of two curved -helices, termed helix-N (Val3-Val37) and helix-C (Lys45-Thr92), connected by a short linker in an anti-parallel arrangement, followed by a short extended region (Gly93-Lys97) and a predominantly unstructured mobile tail (Asp98-Ala140; Fig. 4). Thus, helix-N essentially starts with the beginning of the aS sequence, before the first imperfect repeat, and ends in the third repeat (Fig. 1). The linker then takes up 7 residues before helix-C commences in repeat IV and extends throughout the remainder of the repeat region and terminates after three further residues (Fig. 1). The helix-helix connector forms an extended conformation with a turn propensity near its midpoint (Fig. 4D). Although it has been known that there is a discontinuity in the helical conformation of micelle-bound aS near Ser42-Thr44 (12, 18), the anti-parallel orientation of the helices before and after this break was unexpected. Some variability in interhelical distances was evident from the PRE data, and the structure calculated is somewhat biased to more remote interhelical distances than the actual average (see "Experimental Procedures"). At a range of interhelical distances, including the calculated one, the side chains emanating from the helices were too far apart to interact with each other across the helices (Fig. 4C). An average of 3.60 ± 0.13 residues make up one turn of helix-N with an average twist angle of 100.1 ± 3.8°; for helix-C these parameters are 3.56 ± 0.16 and 101.2 ± 4.7°. Those parameters are not very sensitive to deviations from linear -helical geometry (44). The diameter of a circle best-fitted to the helix and the bending angle between successive local helix axes calculated for 4 residue stretches are better indicators of helix curvature (44, 45). For helix-N the average circle diameter and bending angle were 153 Å and 10.1 ± 5.2°, respectively, whereas for helix-C values of 82 Å and 10.7 ± 6.8°, respectively, were obtained. A slight kink near Val66-Gly67-Gly68, which "incorporates" curvature, appears partly responsible for the stronger bending of helix-C and allows the other stretches of helix-C to wrap around the micelle more intimately (Fig. 4, C–E).
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What structural fluctuations correspond to the dynamic events experienced by aS? To obtain parameters that describe the structure adopted by aS, the chemical shift changes of the backbone HN, N, and C nuclei between the free and micelle-bound states of aS were computed. Each nucleus has different susceptibilities to structural parameters. C chemical shifts depend largely on the structural environment (average backbone dihedral angles), whereas HN and N shifts do so to a much lesser extent and are also strongly influenced by the chemical environment, side-chain conformation and, in the case of HN nuclei, hydrogen bonding (49, 50). The dynamics on the fast (nanosecond to picosecond) and slow (millisecond to second) timescales correlated well with the deviation of the C shifts from their random-coil positions (Fig. 6A). The correlation with the HN shift changes are similar, indicating hydrogen bond formation, whereas for N no clear correlation was discernible (Supplementary Fig. 6). It appears that, for the two helices, the dynamic events on the fast and slow timescales reflect the local helical content. For example, the time fraction over which residues Ala30-Val37 are -helical is considerably diminished as judged by the 50% smaller C secondary shifts and the enhanced slow and fast timescale dynamics compared with the other helical regions. It is therefore not surprising that the magnitude of the RDCs of this region, i.e. of the alignment, is greatly diminished relative to its preceding helical region, or helix-C within Glu83-Ala89. A static structure for this stretch of helix-N does not reflect the actual dynamic events taking place, but merely describes the effective average conformation of the molecule. However, the elevated dynamics on the microsecond timescale observed for the Lys45-Ala56 region was somewhat surprising given its high S2 values and large C secondary shifts, which were comparable to regions with low microsecond timescale dynamics. It follows that, for this region, helical conformation must be maintained throughout any dynamic events taking place. Rather than helix fraying, which will contribute to the large amplitude angular fluctuations of Ala30-Val37, motions in Lys45-Ala56 must involve larger units of well structured helix, i.e. global rather than local motions.
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Structural Basis for the Binding of the Negatively Charged Micelle Surface—To illustrate the interaction of aS with the micelle surface and to examine possible site-specific differences, the electrostatic surface potential of micelle-bound aS has been evaluated (Fig. 7). The inner surface of the curved helices is largely apolar. Most of the basic side chains point sideward from the helices, and the acidic side chains predominantly line their outer surface. To interact with the micelle surface the inner surface of the helices is prone to move past the charged headgroups and interact with the apolar detergent or lipid tails. Concomitantly, the sideward oriented positively charged side chains can engage in interactions with the negatively charged headgroups and thus explain the preference of aS-vesicle interactions on the presence of lipids with negatively charged headgroups (10). Effectively, aS absorbs on the micelle surface and alters its surface charge density, a mode of interaction that classifies it as a hydrophobic cation (51). The helix-helix connector exhibited, with Leu38, Tyr39, Val40, Lys43, and bordering Lys45, a particularly pronounced hydrophobic cationic character (Fig. 7B), which, in turn, may explain its relatively high degree of backbone order on the sub-nanosecond timescale (Fig. 5A).
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70 SDS molecules is expected to be 46 Å (52). However, helix-C appeared to encompass a particle with a much larger diameter of 82 Å (Fig. 4, D and E), indicating the desire to form a less curved helix than a globular SDS micelle would support. Is the native, globular micelle shape deformed by the interaction with aS or do the pronounced microsecond timescale dynamics at the end and beginning of helix-N and -C, respectively, indicate their only transient interaction with the micelle surface? In the presence of hydrophobic cations it has been shown that anionic SDS micelles undergo uniaxial growth from globular micelles to prolate ellipsoidal structures due to a decrease of the electrostatic repulsion of headgroups (53). Thus, it appears that a deformation of the globular micelle along the helix axes to an ellipsoid particle can account for the observed diameter of helix-C. In addition to a deformation of the micelle shape, slight differences in the immersion depth of individual helix regions can also contribute to the observed helix diameter. For Ala30-Val37 of helix-N the helical content is greatly diminished as judged by the very small secondary 13C shifts (Fig. 6A), the rapid backbone amide exchange rates (Fig. 5B), and the high degree of sub-nanosecond timescale dynamics (Fig. 5A) compared with the other helical regions of aS. These characteristics of Ala30-Val37 may contribute to the larger apparent diameter of curvature for helix-N (153 Å) compared with helix-C, in addition to its shorter length than helix-C.
Finally, given that the helices N and C are at variable, relatively close distances and the charges of the basic side chains are shielded by the negatively charged SDS headgroups, an attempt was made to locate short range interproton distances between Val-1,2, Leu-1,2, and Ile-1 methyl groups across the helices using an isotope labeling procedure where these methyl groups were protonated, with the remainder of the protein deuterated (23). Although none could be detected (data not shown), the nonspecific nature of hydrophobic contacts and distance variation between the helices would make such contacts particularly difficult to detect. In addition, the rotameric states of most side chains were found to be averaged (data not shown).
The aS Tail Region Exhibits Non-uniform Properties—Despite the highly mobile nature of the tail region of aS (Fig. 5, A and B), residual dipolar couplings (RDCs) of quite large values were obtained for this region. Denatured or unfolded proteins generally exhibit non-zero RDCs (54), which can be attributed to the differential steric obstruction in anisotropic media of the many conformers that contribute to the random coil behavior (55). In the case of aS, an interesting distribution of the RDCs of the highly mobile tail region was observed. Up to Pro120, RDCs of considerable magnitude were seen, whereas for the remaining tail substantially smaller RDCs were found (Fig. 8A). The larger RDCs for the first compared with the second region suggest a difference in shape of the conformational ensembles of these two segments of the tail (55). The positive sign of the N-H RDC values in the C-terminal tail (Fig. 8A) reflects that the most ordered conformers of the random coil ensemble have their backbone ordered in a direction parallel to the magnetic field, i.e. parallel to the direction in which the gel matrix has been stretched. Finally, small chemical shift changes were induced in the tail by the presence of SDS (Figs. 2B and 8B), which decrease toward the C terminus, suggesting that the changes do not merely arise from the presence of SDS micelles themselves, but also from the structuring of the repeat region.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helices, termed helix-N (Val3-Val37) and helix-C (Lys45-Thr92), connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97) and a predominantly unstructured tail (Asp98-Ala140). The chemical makeup of the applied micelle system (SDS) was found to have little effect on aS structure, as judged by the similarity of aS chemical shifts in the presence SDS-only micelles and mixed micelles composed of DPC and SDS (70:30 molar ratio). The physiological aS target, presynaptic vesicles, exhibit diameters on the order of 50 nm (56), which is about ten times larger than that of an unperturbed SDS micelle. Consequently, the structural implications arising from the less curved and larger surface available to aS in the vesicle-bound state have to be evaluated. An altered helix curvature in the vesicle, compared with the micelle-bound state, will have little effect on the number of residues per turn and helix twist (44). In other words, the local structure encoded by the aS amino acid sequence, which places side chains involved in favorable interactions with the vesicle surface on one side of the helix, remains the same. This is also verified experimentally; for Thr59-Ala90 the side chains lining the inner side of helix-C also face the surface of negatively charged SUV of 300–400 Å diameter (19). Thus, the local structure of helices N and C closely resembles that of the vesicle-bound state. The well ordered, mostly extended helix-helix connector (Leu38-Thr44) is an interesting feature of the micelle-bound structure. Although the presence of a helix break in the micelle-bound state has been suggested to be a consequence of the small size of the micelle (19), the well ordered helix-helix connector detected here indicates a defined interaction with lipidic surfaces and, hence, corresponds to a stable, low energy conformation. Intriguingly, it is conceivable that Leu38-Thr44 could switch between the conformation we observe in the micelle environment and the previously proposed uninterrupted helix when bound to SUV (19) in an environment-dependent manner. Actual presynaptic vesicles are in a complex arrangement with various proteins (57) and, hence, comprise varying environments, which are subject to change during vesicle fusion. An interconversion of the seven Leu38-Thr44 residues between the two conformations would leave helices N and C essentially unperturbed. The short extended stretch following helix-C (Gly93-Lys97) overlaps with the chaperone-mediated autophagy recognition motif of aS (Val95-Gln99), which may implicate impaired aS degradation in the pathogenesis of Parkinson's disease (58). Lastly, the aS tail presents itself mainly unstructured, but small shift changes are induced in the tail by the structuring of the repeat region. This observation is complementary to a recent study of aS-polyamine complexes, which concluded that the tail acts as a regulator of aS aggregation (7). In this study polyamines were found to bind Gln109-Ala140, but induce chemical shift changes also in Thr22-Gly93. The binding of aS to vesicles not only leads to a structuring of aS, but may also impact the stability and membrane fluidity of the vesicle itself. Local differences in structure and dynamics along the helices are therefore of potential importance to aS function. Fast timescale dynamics (sub-nanosecond), which describe fluctuation of individual bond vectors, identify three regions of reduced backbone order, Ala30-Val37, Asn65-Val70, and Glu83-Ala89. Common to all three regions are two Gly residues in close sequential proximity (Fig. 1). Only repeat IV maintains relatively low sub-nanosecond dynamics despite its two proximate glycines. For helices of globular proteins, the 14.7% Gly content of the aS helices would be exceptionally high, but also for integral membrane proteins it is quite high (59). This suggests that the aS glycines serve a functional role. By conferring elevated dynamics within the aS helix, Gly residues may serve to mitigate a possible reduction in membrane fluidity of aS-coated vesicles. In studies of model membranes the segmental motion of spin-labeled lipids was found to be hindered by the presence of aS (60), and a thermodynamic study of aS-SUV interactions indicated lipid ordering upon aS binding (61). In whole cell membrane preparations of neurons, aS-dependent changes in membrane fluidity were observed (62), whereas for isolated synaptosomal membranes no change in membrane fluidity was detected (63). As a common theme of the numerous biophysical studies on aS, including the present one, an impact of aS on vesicle membrane fluidity and stability is increasingly evident. Consequently, the structure-function relationship points toward a modulation of the fusion propensity of presynaptic vesicles by the presence of aS.
The curved helices N and C exhibit average circle diameters of 153 and 82 Å, respectively, which are considerably larger than the expected diameter of a globular SDS micelle of 46 Å. This can be taken as strong evidence for interactions of the positively charged Lys side chains emanating sideward from the helices with the negatively charged headgroups of SDS, leading to a deformation of the globular micelle along the helix axes to a prolate, ellipsoid particle. This mode of interaction has been proposed for apolipoproteins (64) and seems effective in specifying interactions with negatively charged lipidic surfaces and the targeting of specific membranes. At this point it is also noted that a certain degree of curvature is preferred by the aS helix (10), which has been related to the thermodynamics of the aS-vesicle interaction (61). The two helices exhibit variability in interhelical distances and, in particular the end and beginning of helices N and C, respectively, experience unusually large dynamics on the microsecond timescale, which report on the motions of entire stretches of helix, suggesting that interactions with presynaptic vesicle surfaces will be "fluid."
So far the discussion has focused on aspects of membrane binding relevant to aS function. Are any features of aS membrane binding relevant to understanding aS misfolding? Lipid environments that induce -helical conformation have been documented to promote aS aggregation into dimers and multimers (14, 21, 22). Here identified features may therefore be relevant for the initiation of aS misfolding. The region Ala30-Val37 is of prime interest, especially given that the A30P substitution, which causes familial Parkinson's disease (65), localizes in this region. It experiences large amplitude dynamics on all timescales, it exhibits reduced helical content and, excepting its two Gly residues, it is identical in sequence to the well behaved Ala56-Val63 region (Fig. 1). Glu57 and Gln62 correspond to Gly31 and Gly36, and inspection of the structure shows that their side chains do not contact the micelle surface. Taken together, the reduced stability of helical conformation in this region may promote the initial aggregation of aS in non-helical conformation (20).
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S6.
To whom correspondence should be addressed. Tel.: 301-594-8301; Fax: 301-496-0825; E-mail: tobias.ulmer{at}nih.gov.
1 The abbreviations used are: aS, human -synuclein; EPR, electron paramagnetic resonance; PRE, paramagnetic relaxation enhancement; RDC, residual dipolar coupling; SUV, small unilamellar vesicle; TROSY, transverse relaxation optimized spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; TEMED, N,N,N',N'-tetramethylethylenediamine; HSQC, heteronuclear single quantum coherence; CT, constant time.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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58:1, and data points are represented by circles; below this ratio complex exchange behavior was observed and data points are shown as squares. For several residues (e.g. Thr54 and Ala56) a doubling of resonances is observed at an intermediate SDS:aS ratio of 43:1. For Ala29 and Thr54, the chemical shift in the absence of SDS is far off the curve toward the final micelle-bound state. At low SDS:aS ratios extensive resonance broadening prevented the identification of chemical shifts for residues of the aS repeat region (
