Dual Labeling of the CBP/p300 KIX Domain for 19F NMR Leads to Identification of a New Small‐Molecule Binding Site

Protein‐Observed Fluorine NMR (PrOF NMR) spectroscopy is an emerging technique for screening and characterizing small‐molecule–protein interactions. The choice of which amino acid to label for PrOF NMR can be critical for analysis. Here we report the first use of a protein containing two different fluoroaromatic amino acids for NMR studies. Using the KIX domain of the CBP/p300 as a model system, we examine ligand binding of several small‐molecule compounds elaborated from our previous fragment screen and identify a new ligand binding site distinct from those used by native transcription factors. This site was further supported by computational modeling (FTMap and Schrödinger) and 1H,15N HSQC/HMQC NMR spectroscopy. Metabolic labeling with multiple fluorinated amino acids provides useful probes for further studying ligand binding and has led to new insight for allosterically regulating transcription‐factor protein interactions with small‐molecule ligands. Protein‐Observed Fluorine NMR (PrOF NMR) spectroscopy is valuable for characterizing small‐molecule–protein interactions. Here we show how metabolically labeling the KIX domain of CBP/p300 with two different fluorinated amino acids provides better surface coverage for characterizing protein–ligand interactions and has led to identification of a new small‐molecule ligand binding site.