title: Molecular basis for preventing α-synuclein aggregation by a molecular tweezer.
abstract: Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.
authors.affiliation: Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48823.
authors.forename: Srabasti
authors.initials: S
authors.lastname: Acharya
authors.affiliation: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Safaie
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095.
authors.forename: Piriya
authors.initials: P
authors.lastname: Wongkongkathep
authors.affiliation: Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California 90095.
authors.forename: Magdalena I
authors.initials: MI
authors.lastname: Ivanova
authors.affiliation: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095; Brain Research Institute, University of California at Los Angeles, Los Angeles, California 90095.
authors.forename: Aida
authors.initials: A
authors.lastname: Attar
authors.affiliation: Institute of Organic Chemistry and Center for Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany.
authors.forename: Frank-Gerrit
authors.initials: FG
authors.lastname: Klärner
authors.affiliation: Institute of Organic Chemistry and Center for Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schrader
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095; Department of Biological Chemistry, University of California at Los Angeles, Los Angeles, California 90095; Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.affiliation: Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095; Brain Research Institute, University of California at Los Angeles, Los Angeles, California 90095; Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095.
authors.forename: Gal
authors.initials: G
authors.lastname: Bitan
authors.affiliation: Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48823. Electronic address: lapidus@msu.edu.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Lapidus
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001952:Bridged-Ring Compounds / Q000737:chemistry*; D004058:Diffusion; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000737:chemistry*; D010300:Parkinson Disease / Q000378:metabolism; D011336:Probability; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D016896:Treatment Outcome; D051844:alpha-Synuclein / Q000737:chemistry*
chemical_list: D001952:Bridged-Ring Compounds; C583031:CLR01 compound; D010755:Organophosphates; D051844:alpha-Synuclein; D008239:Lysine
keywords: Inhibitor; Intramolecular Diffusion; Mass Spectrometry (MS); Parkinson's Disease; Protein Aggregation; Spectroscopy; α-Synuclein