Point of Contact: Pinpointing Small Molecule-Protein Binding

Understanding how small molecules interact with proteins is crucial in many areas of biology, including enzymology, cell signaling, immunology, and drug development. However, despite their importance, our knowledge of small molecule-protein interactions is still limited, especially when compared to extensively studied protein-protein interactions. Reasons for this gap include technical challenges in capturing small molecule-protein interactions in detail, especially at the level of identifying specific binding sites on the proteins. Several methods exist to identify which proteins interact with a specific small molecule. These include techniques like chemical pull-down proteomics, thermal proteome profiling, and limited proteolysis. While these methods can identify interacting proteins, they often struggle to pinpoint the exact amino acids involved in the interaction. Identifying these precise binding sites is important because it helps differentiate meaningful interactions from background noise and can help predict the biological effects of these interactions. A promising method to map small molecule-protein interactions more precisely involves using photocrosslinking, which freezes transient interactions by using UV light to establish permanent chemical bonds. This technique could, in theory, also reveal the exact binding sites if the crosslinked peptides can be isolated and identified. However, in practice, it is challenging to identify these binding sites confidently due to the low abundance of crosslinks and the unique ways each compound affects the analysis. Similar challenges have been faced in protein-protein interaction studies, but researchers overcame these by using mass-spectrometry with cleavable linkers. These linkers allow crosslinks to be reversed within the mass-spec instrument, leaving behind common, easily identifiable tags that facilitate detecting and analyzing the interactions. The Point of Contact project proposes that combining compound photocrosslinking with mass- spec cleavable linkers can significantly improve our ability to identify the binding sites of small molecules on proteins. We plan to test this approach on three important metabolites: ATP, IMP, and AICAR, which have different levels of prior characterization. If successful, this method could become a standard technique in chemical biology for identifying protein targets and binding sites of small molecules.