Small Molecule Regulation of CLOCK:BMAL1 DNA Binding Activity.

Area of Science:

• Molecular Biology
• Chronobiology
• Biochemistry

Background:

• Circadian rhythms are regulated by transcription factors like CLOCK:BMAL1.
• PAS domains within these factors are known to bind ligands and are potential regulatory sites.
• The CLOCK:BMAL1 complex controls gene expression crucial for biological timing.

Purpose of the Study:

• To investigate if small molecules can bind to CLOCK and NPAS2 PAS domains.
• To determine if ligand binding affects the DNA-binding activity of CLOCK:BMAL1.
• To explore the potential for small molecule-mediated regulation of circadian rhythms.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy was used to identify small molecule ligands.
• A gatekeeping mutant in the CLOCK PAS-A domain was created to assess ligand binding.
• High-pressure NMR studies were performed to evaluate domain stability.
• In vitro assays measured the effect of ligands on CLOCK:BMAL1 DNA binding.

Main Results:

• Small molecules were identified that bind within a cavity in the CLOCK and NPAS2 PAS-A domains.
• A gatekeeping mutation reduced ligand binding affinity.
• Ligand binding and the mutation stabilized the PAS-A domain.
• Ligands induced a dose-dependent displacement of CLOCK:BMAL1 from DNA.

Conclusions:

• Small molecules can bind to specific cavities within the CLOCK:BMAL1 complex's PAS domains.
• Ligand binding to the PAS domain cavity modulates the DNA-binding activity of CLOCK:BMAL1.
• This provides a novel mechanism for regulating circadian transcription factors using small molecules.