Molecular basis for preventing α-synuclein aggregation by a molecular tweezer

Area of Science:

• Biochemistry
• Neuroscience
• Molecular Biology

Background:

• Alpha-synuclein aggregation is a key process in neurodegenerative diseases.
• Aggregation kinetics are influenced by the protein chain's reconfiguration rate.
• Molecular chaperones and small molecules can modulate protein aggregation.

Purpose of the Study:

• To investigate the effect of the molecular tweezer CLR01 on alpha-synuclein aggregation.
• To determine the binding site and mechanism of action of CLR01 on alpha-synuclein.
• To explore how CLR01 influences the kinetic control of alpha-synuclein self-assembly.

Main Methods:

• Fluorescence spectroscopy to measure binding and reconfiguration rates.
• Mass spectrometry (including top-down analysis) to identify binding sites.
• Photo-induced cross-linking of unmodified proteins (PICUP) to assess protein aggregation state.
• Kinetic modeling to interpret experimental results.

Main Results:

• CLR01 binds strongly to multiple lysine residues in alpha-synuclein, primarily Lys-10/Lys-12.
• CLR01 increases the reconfiguration rate of alpha-synuclein in a concentration-dependent manner.
• Under experimental conditions, alpha-synuclein was predominantly monomeric, and CLR01 accelerated monomer reconfiguration.
• Kinetic modeling supported a mechanism where faster reconfiguration disfavors aggregation.

Conclusions:

• CLR01 effectively inhibits alpha-synuclein aggregation by accelerating its reconfiguration rate.
• The N-terminal lysine residues (Lys-10/Lys-12) are key binding sites for CLR01 on alpha-synuclein.
• CLR01's mechanism provides insights into preventing neurotoxic aggregate formation in alpha-synuclein and potentially other amyloidogenic proteins.