Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

Katerina E Paleologou,Adrian W Schmid,Carla C Rospigliosi,Hai-Young Kim,Gonzalo R Lamberto,Ross A Fredenburg,Peter T Lansbury,Claudio O Fernandez,David Eliezer,Markus Zweckstetter,Hilal A Lashuel

doi:10.1074/jbc.m800747200

Abstract

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

Highlights

A study by Fujiwara et al [8] reported that in vitro phosphorylated ␣-syn (at Ser-129, using casein kinase II (CK2)) forms fibrils more readily than unmodified ␣-syn
We considered that a rigorous examination and comparison of the biochemical and biophysical properties of phosphorylation mimicking mutants (S129A and Ser-129 was replaced by Glu (S129E)/D), and WT ␣-syn may clarify the observations described above as well as the molecular mechanisms by which phosphorylation at Ser-129 may modulate ␣-syn aggregation and toxicity in vivo
Important is our finding that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation at this site on ␣-syn structure and aggregation properties in vitro

Summary

EXPERIMENTAL PROCEDURES

Expression, and Purification of ␣-Syn Variants— The S129E, S129D, S129A, and S87A ␣-syn mutants were generated using site-directed mutagenesis employing complementary internal mutagenic primers and two-step PCR. Fibrillation Studies—To probe the effect of CK1 phosphorylation on the aggregation of ␣-syn, WT ␣-syn was phosphorylated for 24 h at 30 °C, and the reaction was stopped with EDTA disodium salt before the samples were subjected to fibrillation conditions at 37 °C with continuous shaking for the indicated time points. The Far UV-CD spectra (190 –250 nm, integration time of 2 s for 0.2 nm) were collected at room temperature in a 1-mm path length quartz cuvette containing 0.1 mg/ml of ␣-syn in PBS or sodium phosphate buffer. Pulse field gradient NMR experiments were acquired on unlabeled WT and mutant ␣-syn (200 ␮M) dissolved in 99.9% D2O, 50 mM phosphate buffer, pH 7.4, and containing dioxane (ϳ20 mM) as an internal radius standard and viscosity probe [21]. Lyophilized S87A, either unphosphorylated or phosphorylated, was dissolved in sample buffer (100 mM NaCl, 10 mM Na2HPO4, pH 7.4, in 90%, 10% H2O, D2O) with 40

G68 P hosphorylated W T

RESULTS

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