Molecular Basis for Preventing α-Synuclein Aggregation by a Molecular Tweezer

• Published in: The Journal of biological chemistry 2014-04, Vol.289 (15), p.10727-10737
• Main Authors: Acharya, Srabasti, Safaie, Brian M., Wongkongkathep, Piriya, Ivanova, Magdalena I., Attar, Aida, Klärner, Frank-Gerrit, Schrader, Thomas, Loo, Joseph A., Bitan, Gal, Lapidus, Lisa J.
• Format: Article
• Language: English
• Subjects: alpha-Synuclein - chemistry; Amino Acid Sequence; Binding Sites; Bridged-Ring Compounds - chemistry; Diffusion; Humans; Inhibitor; Intramolecular Diffusion; Kinetics; Lysine - chemistry; Mass Spectrometry; Mass Spectrometry (MS); Molecular Bases of Disease; Molecular Sequence Data; Organophosphates - chemistry; Parkinson Disease - metabolism; Parkinson's Disease; Probability; Protein Aggregation; Protein Binding; Protein Structure, Tertiary; Spectrometry, Fluorescence; Spectroscopy; Treatment Outcome; α-Synuclein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.524520

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. Background: The molecular tweezer, CLR01, binds to Lys and prevents aggregation of α-synuclein. Results: CLR01 binds directly to monomeric α-synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. Conclusion: Aggregation is inhibited by making the protein more diffusive. Significance: The most effective aggregation inhibitors may change monomer dynamics rather than structure.