ABSTRACT
Protein-Observed Fluorine NMR (PrOFâ NMR) spectroscopy is an emerging technique for screening and characterizing small-molecule-protein interactions. The choice of which amino acid to label for PrOFâ 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 1 H,15 N 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.
Subject(s)
Small Molecule Libraries/pharmacology , p300-CBP Transcription Factors/metabolism , Animals , Binding Sites/drug effects , CREB-Binding Protein/chemistry , CREB-Binding Protein/metabolism , Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Fluorine/analysis , Humans , Mice , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Domains/drug effects , Protein Interaction Maps/drug effects , Rats , Small Molecule Libraries/chemistry , p300-CBP Transcription Factors/chemistryABSTRACT
Thioethers, sulfoxides, and sulfonium ions, despite diverse physicochemical properties, all engage in noncovalent interactions with proteins. Thioether-containing macrocycles are also attracting attention as protein-protein interaction (PPI) inhibitors. Here, we used a model PPI between α-helical mixed lineage leukemia (MLL) protein and kinase-inducible domain interacting (KIX) domain to evaluate oxidation effects on sulfurcontaining macrocycle structure, stability, and protein affinity. Desolvation effects from various polarity states were evaluated computationally and experimentally at the side chain, amino acid, and peptide level. Sulfur-containing side chains spanned polarity ranges between all-hydrocarbon and lactam bridges for modulating solubility, cellular uptake, and affinity. Helical propensity studies showed that, although oxidized sulfur-containing side chains could be tolerated, conformational effects were sequence-dependent. In some cases, proteolytic stability, binding capacity with KIX, and increased helicity were obtained as first steps toward developing PPI inhibitors.
