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

Protein-Observed Fluorine NMR (PrOFNMR) spectroscopy is an emerging technique for screening and characterizing small-molecule-protein interactions. The choice of which amino acid to label for PrOFNMR 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 Schrodinger) and H-1,N-15 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.