Calcium-Triggered Membrane Interaction of the R-Synuclein Acidic Tail

R-Synuclein ( R-syn) is a 140-residue protein that aggregates in intraneuronal inclusions called Lewy bodies in Parkinson’s disease (PD). It is composed of an N-terminal domain with a propensity to bind lipids and a C-terminal domain rich in acidic residues (the acidic tail). The objective of this study was to examine the effect of Ca 2+ on the acidic tail conformation in lipid-bound R-syn. We exploit the extreme sensitivity of the band III fluorescence emission peak of the pyrene fluorophore to the polarity of its microenvironment to monitor subtle conformational response of the R-syn acidic tail to Ca 2+. Using recombinant humanR-syn bearing a pyrene to probe either the N-terminal domain or the acidic tail, we noted that lipid binding resulted in an increase in band III emission intensity in the pyrene probe tagging the N-terminal domain but not that in the acidic tail. This suggests that the protein is anchored to the lipid surface via the N-terminal domain. However, addition of Ca 2+ caused an increase in band III emission intensity in the pyrene tagging the acidic tail, with a corresponding increased susceptibility to quenching by quenchers located in the lipid milieu, indicative of lipid interaction of this domain. Taken together with the increasedâ-sheet content of membrane-associated R-syn in the presence of Ca 2+, we propose a model wherein initial lipid interaction occurs via the N-terminal domain, followed by a Ca 2+-triggered membrane association of the acidic tail as a potential mechanism leading to R-syn aggregation. These observations have direct implications in the role of age-related oxidative stress and the attendant cellular Ca2+ dysregulation as critical factors in R-syn aggregation in PD. R-Synuclein ( R-syn)1 belongs to a family of small, soluble proteins called synucleins ( R-, â-, andγ-synuclein) localized predominantly in neuronal tissues and to a lesser extent in hematopoietic cells and platelets ( 1, 2). It plays a key role in Parkinson’s disease (PD), a neurodegenerative disorder characterized by dramatic losses of dopaminergic neurons in the substantia nigra and in other dementias such as dementia with Lewy body. A hallmark pathological feature of PD is the occurrence of intraneuronal inclusions called Lewy bodies, which are intensely eosinophilic ( 3). The Lewy bodies are composed predominantly of R-syn and lipids, the former deposited as high molecular weight aggregates in a fibrillar state, suggesting loss in solubility or increased protein-protein interactions as putative reasons for aggregation. Two autosomal point mutations in R-syn (A53T and A30P) and overexpression of the wild-type (WT) R-syn pose a high risk for developing PD, although they account for a mere 1% of all PD cases ( 4, 6); aging appears to be a consistent risk factor in the onset of PD, and there is a consensus about the crucial role for this protein in the pathology of PD, though the exact structural and functional mechanisms of action are not understood. R-Syn is composed of 140 residues (Figure 1A) and structurally characterized by three modular domains: (a) an N-terminal domain with seven conserved imperfect 11residue repeats, bearing a propensity to form amphipathic R-helices and to interact with lipid surfaces ( 7), (b) a â-amyloid binding domain encoding the non-Abeta component (NAC) of Alzheimer’s disease plaques, and (c) a C-terminal tail, characterized by an abundance of negatively charged residues, which binds, among other factors, Ca 2+ and microtubule-associated proteins 1B ( 8, 9). Interestingly, lipid binding ofR-syn is mediated via the N-terminal segment, which elicits a binding preference for membranes containing negatively charged phospholipids (10), which the weakly anionic C-terminal tail has a natural tendency to repel. Under physiological conditions, R-syn is found in a soluble form in the cytosol and is predominantly disordered ( 11). It is acknowledged that the pathogenesis triggered byR-syn is likely due to structural alterations involving transition from a monomeric unstructured state to † This work was supported by funding from the Parkinson’s Disease Foundation and from the Alzheimer’s Association (TLL-03-5281) to V.N. , from the National Funds for Scientific Research (Belgium) to V.R. , and from the American Heart Association postdoctoral fellowship to S.T. * Address correspondence to this author. Tel: (510) 428-3885 ext 2965. Fax: (510) 450-7910. E-mail: vnarayan@chori.org. ‡ Children’s Hospital Oakland Research Institute. § UniversitéLibre de Bruxelles. 1 Abbreviations:R-syn,R-synuclein; CD, circular dichroism; 5-DSA, 5-DOXYL-stearic acid; IR, infrared; NAC, non-Abeta component; NPM, N-(1-pyrene)maleimide; PD, Parkinson’s disease; POPA, 1-palmitoyl-2-oleoylphosphatidic acid; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; TCEP, tris(2-cyanoethyl)phosphine; TFE, 2,2,2-trifluoroethanol; WT, wild type. 10947 Biochemistry2006,45, 10947-10956 10.1021/bi060939i CCC: $33.50 © 2006 American Chemical Society Published on Web 08/16/2006 a â-sheet filamentous state ( 12), with a partially folded intermediate likely playing a critical role in the aggregation process ( 13). Functionally,R-syn has also been implicated in dopamine and Ca2+ signaling, with the C-terminal segment likely involved in the Ca2+ binding process ( 14, 15). Other putative functions include a role in neurotransmitter release from presynaptic vesicles and modulation of presynaptic vesicle pool size, plasticity, and recycling via the dopamine transporter (16) and other calcium-mediated roles ( 14, 15, 17, 18). A possible clue to the role of R-syn may lie in the fact that aging is consistently associated as a risk factor for PD. Inevitably, oxidative stress appears to be a necessary accompaniment of the normal aging process and is closely associated with the neurobiology of aging, including neurodegeneration in Alzheimer’s disease and PD ( 19). In addition, oxidative stress is intrinsically linked to Ca 2+ dysregulation, with documented reports of increase in Ca 2+ influx and intracellular Ca 2+ leading to a severe disruption of the normal cellular processes ( 20-23). This further implies that the presynaptic terminal, with an abundance of R-syn, lipids, and high local concentration of Ca 2+, is a crucial site for initiating pathogenesis of PD. In this study we propose that Ca 2+ triggers a conformational restructuring of the acidic tail of R-syn that is anchored to the membrane surface via its N-terminal domain. We employ site-directed fluorescence labeling of recombinant humanR-syn to evaluate the relative spatial disposition of its N-terminal and C-terminal segments in lipid-free and lipid-bound states. Our studies suggest that Ca 2+ either bridgesR-syn to the membrane, possibly by coordinating with the negative charge on the R-syn acidic tail and the acidic headgroups in the phospholipid bilayer, or facilitates this segment to traverse the membrane bilayer. MATERIALS AND METHODS Materials. N-(1-Pyrene)maleimide (NPM), tris(2-cyanoethyl)phosphine (TCEP), and N-(1-pyrenesulfonyl)-1,2-hexadecanoylsn-glycero-3-phosphoethanolamine were obtained FIGURE 1: Rationale and design of R-syn constructs for site-directed pyrene labeling. Panel A: Primary structure of R-syn. The amino acid sequence of R-syn (residues 1 -140) is shown wherein single cysteine residues were substituted at position 11 or 124 in lieu of Ala (red), providing sites for covalent attachment of pyrene tags. The C-terminal tail of R-syn comprises a novel tandem repeat of 16 residues ( 14) between positions 109 and 140 that bear acidic residues (green) at defined intervals. Panel B: Microenvironment-sensitive spectral features of pyrene. Fluorescence emission spectra of free pyrene in (i) aqueous buffer (10 mM HEPES, pH 7.4), (ii) acetone, and, (iii) n-hexane. The dielectric constants for these solvents are 80.4, 20.7, and 1.9, respectively, at 20 °C; arrows draw attention to the band III peak at ∼387 nm. Fluorescence emission spectra of free pyrene were recorded in various solvents as indicated, and the band I/III ratio was plotted versus the dielectric constant of the solvents (iv). The spectra were recorded at an excitation wavelength of 340 nm. 10948 Biochemistry, Vol. 45, No. 36, 2006 Tamamizu-Kato et al.