Direct Evidence of Coexisting Horseshoe and Extended Helix Conformations of Membrane-Bound Alphy-Synuclein

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Direct Evidence of Coexisting Horseshoe and Extended Helix Conformations of Membrane-Bound Alphy-Synuclein Direct Evidence of Coexisting Horseshoe and Extended Helix Conformations of Membrane-Bound Alpha-Synuclein Marta Robotta}bl Patrick Braun}bl Bart van Rooijen}cl Vinod Subramaniam,*[cl Martina Huber,*[al and Malte Drescher*[bl a-Synuclein (as) is a 140-residue protein abundantly present in the Lewy bodies characteristic of Parkinson's disease.[1-31 It is a member of the class of intrinsically disordered proteins (lOPs) that have unusual properties and whose physiological rele­ vance is becoming increasingly recognized.[41 lOPs lack a well­ defined three-dimensional fold and display remarkable confor­ mational flexibility. This property potentially enables them to be promiscuous in their interactions and to adapt their struc­ ture according to the needed function. In the case of as, the Figure 1. NMR structure!13! of micelle bound as (bottom) and hypothetical model of an extended a helix (top). Spin label positions are indicated in red. protein is capable of adopting a ~-sheet structure in the amy­ loid fibrils constituting the Lewy bodies and an a-helical struc­ (1 71 ture in the membrane bound form. The exact physiological ameter 5 nm ) used in these studies may have artificially con­ role of as has yet to be determined, but membrane binding strained the protein into a horseshoe structure, and subse­ seems to be important for its function.[S-81 As a consequence, quent studies were performed on small unilamellar vesicles the membrane bound form has received considerable atten­ (SUVs) and bicelles. Of the eight studies published on this tion in the last several years. issue to date, three support the horseshoe[18-201 conformation, It is generally accepted that upon binding to membranes as and five the extended[s. 21-241 conformation, despite using simi­ adopts an amphipathic, a-helical structure involving residues lar experimental techniques and often with only slight varia­ 1- 100.[9-151 The exact arrangement of the helix on the mem­ tions in experimental conditions. There can be several reasons brane surface is, however, controversial. The NMR structure of for the apparent discrepancies. The protein could be sensitive as bound to SOS micelles revealed a break in the helix, result­ to the exact experimental conditions, accessible distance ing in two anti parallel alpha-helices (horseshoe model, ranges of the method could limit the observation to only one Figure 1, bottom).[131 This model was confirmed by techniques of the forms and incomplete binding could aggravate the un­ based on electron paramagnetic resonance (EPR).[161 It has certainty. been postulated that the small size of the micelles (typical di- Herein we present evidence that the horseshoe configura­ tion is also found on larger vesicles, and that under these con­ [a] Dr. M. Huber ditions as coexists in a superposition of both horseshoe and Leiden Institute of Physics extended forms shown in Figure 1. University of Leiden The vesicles used herein were large unilamellar vesicles (LUV, PO Box 9504, 2300 RA Leiden (The Netherlands) 100 nm in diameter) of the negatively charged lipid POPG Fax: (+ 31)71 5275819 Email: mhuber@malphys.leidenuniv.nl {l -Palmitoyl-2-0Ieoyl-sn-Glycero-3-[Phosphorac-(l -glycerol)]}. It Homepage: www.molphys.leidenuniv.nllmonosl has been shown that as binds most effectively to negatively [b] M. Robotta, + P. Braun, + Dr. M. Drescher charged membranes consistent with the exposure of positively Department of Chemistry charged amino acid residues in the helical section of the N-ter­ University of Konstanz minus of as.[131 The POPG LUVs are stable upon addition of as Universitiitsstr. 10, 78457 Konstanz (Germany) Fax: (+ 49)753 88 3139 and continuous wave (cw) EPR on singly labelled as, according Email: malte.drescher@unikonstanz.de to an approach described before,[201 which showed that, under Homepage: www.unikonstanz.deldrescher the conditions used here, quantitative binding is achieved. [c] Dr. B. van Rooijen, Prof. Dr. V. Subramaniam Quantitative binding was also confirmed by fluorescence corre­ Nanabiophysics, MESA + lation spectroscopy (FCS) [Supporting Information]. Further­ Institute for Nanotechnology & MIRA Institute for Biomedical Technology and Technical Medicine more, no evidence for aggregates or oligomers of as on the University of Twente membrane, such as those observed for as on POPG SUVSPSI PO Box 217, 7500AE Enschede (The Netherlands) was found under the conditions of the present investigation Fax: (+ 31)53 4891105 (Supporting Information). Email: v.subramaniam @utwente.nl Homepage: www.utwente.nllnbp In order to monitor the structure of membrane-bound as, a [+] These authors contributed equally to this work. set of double cysteine mutants was specifically labelled with Q Supporting information for this article is available on the WWW under MTSL [l-oxyl-(2,2,S,S-tetra methylpyrroli ne-3-methyl)methan e­ http://dx.doLorgIl0.1002/cphc.201000815. thiosulfonatel, using the site-directed spin labelling approach 267 pioneered by Hubbell et a1.126) Spin labels were attached at po­ A) 8) 1.0 ( : 4.3 nm sitions 9 and 69 (aS9/69), aS9/90, aS18/69, aS18/90, and aS27/56 as indicated in Figure 1. Distances between the spin t labels were obtained by the pulsed, two-frequency EPR 0.8 method known as double electron- electron resonance §: :.. 1;;' (DEER},127) which, in principle, provides access to distances be­ c- it :;;: tween 1.5 nm and 8 nm.128) Distances below 1.5 nm were ex­ cluded by cw EPR for all double mutants (Supporting Informa­ tion). The DEER data were analyzed (Supporting Information) 0.4 , to extract the model-free distance distributions. For aS9/69, "-,--, aS9/90, as 18/69, and as 18/90 these distributions are well de­ 0.0 0 ..2 0.4 0,6 0.8 1.0 1.2 1.4 6 1 tI ~s scribed by a single Gaussian, the parameters of which are r l nm given in Table 1. The width of the distribution results from the Figure 2. A) DEER time trace after background correction for u S27156 flexibility of the spin-label linker as well as from the conforma­ bound to POPG LUV (black) and fit (blue) corresponding to the distance dis tional flexibility of the protein. The distances obtained for tribution shown in: B) Distance distribution derived model free by Tikhonov regulari sa tion (black) described by two Gaussians (green). these four mutants are in good agreement with the horseshoe model. The extended conformation should give distances of 8 nm or larger. the two Gaussian distance distributions combine to almost In contrast to the other mutants, the DEER time trace (Fig­ 100%. Thus it is only for this mutant, for which two distances ure 2A) for the aS27/56 mutant cannot be described by a ,are observed and attributed to the horseshoe and extended single Gaussian. A model-free analysis using l1khonov regulari ­ forms that the modulation depth accounts for all protein pres­ sation results in the distance distribution depicted in Figure 2 B ent. We therefore conclude that for the first four mutants a sig ­ (black line), which clearly consists of two contributions (see the nificant fraction of the proteins are in the extended form,ls;22-24) Supporting Information for the L-curve). This model-free dis­ and therefore escape detection. The amount of these "missing tance distribution is well described by two Gaussians, and fits spins" is notoriously difficult to quantify. In addition to the pro­ of the DEER time traces with a superposition of two Gaussian teins in the extended form, that is, with a distance larger than distance distributions lead to similar final parameters (Support­ the measurement range, this population also contains proteins ing Information), confirming that the analysis is model-inde­ that have only one functional spin label and possibly as that is pendent. The shorter-distance Gaussian agrees well with the not bound to the membrane (Supporting Information). Even expected distance of 2.7 nm for the horseshoe conformation under our conditions, an uncertainty of 15 % in these values derived from the NMR structure (pdb access code 1XQ8}113) cannot be avoided (Supporting Information), underlining the while the longer-distance Gaussian is consistent with the need to measure both conformations, which is only possible 4.9 nm expected for an extended a-helix. This can only be ex­ for the aS27/56 mutant in our case. plained if horseshoe and extended forms coexist under the The presence of the horseshoe conformation on the LUVs membrane conditions employed herein. revealed by all five mutants investigated herein clearly shows To determine how this observation ties in with the results that this conformation is stable on intact large vesicles, and is on the remaining mutants, we analyzed the fraction of spin therefore not likely to be an artifact induced by the small size pairs, which is obtained from the modulation depth of the of SUVs or micelles. Consequently it can be considered a phys­ DEER time trace. The fraction of spins obtained from mutants iologically relevant structure on two-dimensional membranes. aS9/69, aS9/90, as 18/69, and as 18/90 (Supporting Informa­ The fraction of the horseshoe conformation is significantly tion) is significantly smaller than 100%, whereas for aS27/56 smaller than that of the extended conformation, however, which reveals a preference for the extended form. We have previously speculated that the Table 1. Paramete;~ of Gaussian distance distributions for doubly labeled as derivatives bound to POPG LUVs horseshoe conformation could obtained by DEER and distances expected from NMR structure" 31 and model of extended
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