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

Srabasti Acharya,Brian M Safaie,Piriya Wongkongkathep,Magdalena I Ivanova,Aida Attar,Frank-Gerrit Klärner,Thomas Schrader,Joseph A Loo,Gal Bitan,Lisa J Lapidus

doi:10.1074/jbc.m113.524520

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.

Highlights

The molecular tweezer, CLR01, binds to Lys and prevents aggregation of ␣-synuclein
We show that the molecular tweezer, CLR01, known to bind predominantly to Lys side chains, behaves to curcumin in increasing protein reconfiguration and
The introduction of the Trp allows using UV fluorescence to investigate the binding of the molecular tweezer by its effect on the quantum yield and maximum emission wavelength, which in turn reveals information about the protein conformation

Summary

Background

The molecular tweezer, CLR01, binds to Lys and prevents aggregation of ␣-synuclein. Results: CLR01 binds directly to monomeric ␣-synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. A potential therapeutic strategy to prevent PD is to use small molecules that bind to ␣-synuclein and increase its reconfiguration rate under physiological conditions, thereby preventing aggregation This strategy was demonstrated with curcumin, a naturally occurring compound in the spice turmeric, which was found to bind strongly to ␣-synuclein, prevent fibrilization, and increase the rate of protein reconfiguration at physiological temperatures [8]. We show that the molecular tweezer, CLR01, known to bind predominantly to Lys side chains, behaves to curcumin in increasing protein reconfiguration and. Fluorescence changes show that increasing CLR01 concentration slows down the early oligomerization process These results suggest that reversing the net charge on certain residues from positive to negative modulates intramolecular interactions in a manner that prevents collapse and speeds up reconfiguration. The data suggest a possible roadmap for therapeutic development

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION