Protocol to identify drug-binding sites in proteins using solution NMR spectroscopy

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Dusquetide is a novel innate defense regulator (IDR) that modulates innate immune responses.
• It targets SQSTM1/p62, a key autophagy receptor protein, suggesting a role in cellular regulation.
• Understanding the precise binding interaction is crucial for its therapeutic development.

Purpose of the Study:

• To establish a detailed protocol for identifying dusquetide-binding sites on the p62 protein.
• To utilize solution Nuclear Magnetic Resonance (NMR) spectroscopy for this characterization.
• To provide a method applicable to other small molecules interacting with p62's ZZ domain.

Main Methods:

• Sample preparation of p62 for NMR analysis.
• Setting up and executing solution NMR experiments.
• Processing NMR data and performing binding site analysis.

Main Results:

• A robust protocol for determining dusquetide-binding sites on p62 was successfully developed.
• The methodology leverages solution NMR spectroscopy for high-resolution interaction mapping.
• The protocol is adaptable for studying other ligands targeting the p62 ZZ domain.

Conclusions:

• The described NMR protocol provides a reliable method for characterizing small molecule interactions with p62.
• This technique facilitates a deeper understanding of dusquetide's mechanism of action.
• The protocol's versatility extends to the study of other ZZ domain-containing proteins.