Characterization of inhibitor-bound alpha-synuclein dimer: role of alpha-synuclein N-terminal region in dimerization and inhibitor binding.
alpha-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other alpha-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit alpha-synuclein assembly with low micromolar IC(50) values, and that SDS-stable, noncytotoxic soluble alpha-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound alpha-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound alpha-synucleins in detail, we have prepared alpha-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated alpha-synuclein monomer and dimer were oxidized at all four methionine residues of alpha-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of alpha-synuclein. Ultra-high-field NMR analysis of inhibitor-bound alpha-synuclein dimers showed that the signals derived from the N-terminal region of alpha-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric alpha-synuclein. We propose that the N-terminal region of alpha-synuclein plays a key role in the formation of alpha-synuclein assemblies.
Yamaguchi
Yoshiki
Y
Department of Structural Biology and Biomolecular Engineering, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. yyoshiki@riken.jp
Masuda
Masami
M
Sasakawa
Hiroaki
H
Nonaka
Takashi
T
Hanashima
Shinya
S
Hisanaga
Shin-ichi
S
Kato
Koichi
K
Hasegawa
Masato
M
D000595:Amino Acid Sequence; D001577:Benzophenones / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D004298:Dopamine / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051844:alpha-Synuclein / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001577:Benzophenones; D005419:Flavonoids; D011994:Recombinant Proteins; D051844:alpha-Synuclein; C052932:exifone; C059922:gossypetin; D004298:Dopamine