Familial Mutants of α-Synuclein with Increased Neurotoxicity Have a Destabilized Conformation

A30P and A53T mutations of the presynaptic proteinα-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of α-synuclein. However, α-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of α-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors. A30P and A53T mutations of the presynaptic proteinα-synuclein are associated with familial forms of Parkinson disease. NMR spectroscopy demonstrates that Parkinsonism-linked mutations greatly perturb specific tertiary interactions essential for the native state of α-synuclein. However, α-synuclein is not completely unfolded but exhibits structural fluctuations on the time scale of secondary structure formation and loses its native conformation gradually when protein stability decreases. The redistribution of the ensemble of α-synuclein conformers may underlie toxic gain-of-function by fostering self-association and altered binding affinity to ligands and receptors. Parkinson disease (PD) 1The abbreviations used are: PD, Parkinson disease; αS, α-synuclein; RDC, residual dipolar coupling; wt, wild type; NAC, non-Aβ component of Alzheimer disease amyloid; PRE, paramagnetic relaxation enhancement; MTSL, 1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl methanethiosulfonate; HSQC, heteronuclear single quantum coherence. is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra and the deposition of eosinophilic fibrillar proteinaceous aggregates known as Lewy bodies (1Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1099) Google Scholar). A central role in this neurological disorder is played by the abundant, presynaptic 140-residue proteinα-synuclein (αS). αS is the major component of Lewy bodies (2Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6177) Google Scholar), and locus triplication causing an increased dosage of the wild type (wt) αS gene potentiates PD (3Singleton A.B. Farrer M. Johnson J. Singleton A. Hague S. Kachergus J. Hulihan M. Peuralinna T. Dutra A. Nussbaum R. Lincoln S. Crawley A. Hanson M. Maraganore D. Adler C. Cookson M.R. Muenter M. Baptista M. Miller D. Blancato J. Hardy J. Gwinn-Hardy K. Science. 2003; 302: 841Crossref PubMed Scopus (3510) Google Scholar). Two autosomal dominantly inherited forms of PD are caused by Ala53 to Thr (A53T) and Ala30 to Pro (A30P) mutations in the αS gene, which were identified in Italian (4Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6667) Google Scholar) and German kindreds (5Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3321) Google Scholar), respectively. Both αS mutants display an impaired degradation by chaperone-mediated autophagy (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1572) Google Scholar) and share an increased tendency to form soluble oligomeric intermediates, with the A53T mutant fibrillating even faster than the wt protein (7Conway K.A. Lee S.J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Crossref PubMed Scopus (1341) Google Scholar). Recent reports suggest that these soluble oligomers, or protofibrils, may constitute the major toxic species, whereas mature fibrils may account for a protective function (8Volles M.J. Lansbury P.T. Biochemistry. 2003; 42: 7871-7878Crossref PubMed Scopus (405) Google Scholar). As the loss of αS appears to have minimal effects on development (9Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M.G. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), the pathogenic effects of mutant αS are attributed to a toxic gain-offunction. These findings are intriguing inasmuch as αS has generally been considered as an intrinsically unstructured protein (10Weinreb P.H. Zhen W. Poon A.W. Conway K.A. Lansbury Jr., P.T. Biochemistry. 1996; 35: 13709-13715Crossref PubMed Scopus (1321) Google Scholar). Why and how single missense mutations in disordered αS accelerate its oligomerization and contribute to the degeneration of dopamine nerve cells are questions fundamental to the understanding and treatment of PD. Recently, it was shown that native αS adopts an ensemble of conformations that are stabilized by long range interactions (11Hoyer W. Cherny D. Subramaniam V. Jovin T.M. Biochemistry. 2004; 43: 16233-16242Crossref PubMed Scopus (276) Google Scholar, 12Dedmon M.M. Lindorff-Larsen K. Christodoulou J. Vendruscolo M. Dobson C.M. J. Am. Chem. Soc. 2005; 127: 476-477Crossref PubMed Scopus (570) Google Scholar, 13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar, 14Rasia R.M. Bertoncini C.W. Marsh D. Hoyer W. Cherny D. Zweckstetter M. Griesinger C. Jovin T.M. Fernandez C.O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4294-4299Crossref PubMed Scopus (340) Google Scholar). The latter form an intricate network involving the N (residues 1–60) and C termini (residues 109–140), which shields the highly amyloidogenic, non-Aβ component of Alzheimer disease amyloid (NAC) (residues 61–95) from the solvent (Fig. 1A). Polyamine binding and temperature increase release the intrinsic tertiary structure of αS, leading to a completely unfolded conformation (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). The relevance of these conditions, which cause aggregation in vitro, for the development of PD is, however, unclear. On the other hand, very strong support for the importance of αS in PD comes from the A30P and A53T mutations in the αS gene linked to familial forms of the disease. Using NMR residual dipolar couplings (RDCs) and parmaagnetic relaxation enhancement (PRE) we demonstrate here that Parkinsonism-linked mutations greatly perturb the ensemble of α-synuclein conformers. This conformationally altered αS may constitute a common mediator in the induction of PD by both environmental and genetic conditions. Protein Preparation—PT7 plasmids containing wt, A30P, and A53T αS sequences were a gift from the Lansbury Laboratory, Harvard Medical School, Cambridge, MA. The following αS cysteine-containing mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) on the above mentioned plasmids: Ala18Cys/A30P, Ala90Cys/A30P, Ala140Cys/A30P, Ala18Cys/A53T, Ala90Cys/A53T, and Ala140Cys/A53T. The introduced modifications were further verified by DNA sequencing. The positions of the cysteine replacements was distributed along the sequence of αS and their introduction did not cause chemical shift changes in residues other than those surrounding the point of mutation. 15N-labeled αS, wt, and mutants were expressed in Escherichia coli grown in M9 minimal medium supplemented with15NH4Cl (Cambridge Isotope Laboratories) and purified as described previously (15Hoyer W. Antony T. Cherny D. Heim G. Jovin T.M. Subramaniam V. J. Mol. Biol. 2002; 322: 383-393Crossref PubMed Scopus (419) Google Scholar). The final protein working solutions were dialyzed against buffer A (20 mm Tris-HCl buffer, pH 7.4, 100 mm NaCl). Spin Labeling of αS—The nitroxide spin label chosen for reaction with the cysteine-containing mutants was MTSL (1-oxy-2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate (Toronto Research Chemicals, Toronto, Ontario, Canada). MTSL had already proven to efficiently react with αS cysteine mutants, and the reaction was carried out as described previously (13Bertoncini C.W. Jung Y.S. Fernandez C.O. Hoyer W. Griesinger C. Jovin T.M. Zweckstetter M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1430-1435Crossref PubMed Scopus (630) Google Scholar). Paramagnetic samples were measured at 100 μm MTSL-labeled protein concentration, and diamagnetic experiments were performed with 100 μm protein in the presence of 0.5 mm dithiotreithol (Sigma) to counteract undesirable intensity changes in certain amide resonances of αS caused by addition of the oxidant ascorbic acid. Alignment of αS in Anisotropic Media—RDCs were measured in αS variants aligned in 5% w/v n-octyl-penta(ethylene glycol)/octanol (C8E5) (Sigma) (16Rückert M. Otting G. J. Am. Chem. Soc. 2000; 122: 7793-7797Crossref Scopus (540) Google Scholar). Formation of the anisotropic, dilute liquid crystalline phase was monitored by the splitting of the deuterium signal, which was 20 ± 2 Hz. Alternatively, alignment was achieved by addition of 10 mg/ml of Pf1 bacteriophage (Asla, Riga, Latvia) with deuterium splittings of 9 ± 1 Hz (17Hansen M.R. Mueller L. Pardi A. Nat. Struct. Biol. 1998; 5: 1065-1074Crossref PubMed Scopus (690) Google Scholar). NMR Measurements—NMR spectra were acquired at 15 °C on Bruker Avance 600 and 700 NMR spectrometers on a 100 μm sample of αS variants in buffer A. Measurement conditions of low temperat

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