Small molecule interactome mapping by photo-affinity labelling (SIM-PAL) is an unbiased target identification and binding site characterization method that enables direct observation of small molecule–protein interactions by mass spectrometry (MS). In SIM-PAL, a small molecule of interest modified with a diazirine and alkyne tag is incubated with a proteome. Local binding interactions are captured upon photolysis of the diazirine. The captured binding sites are enriched by a cleavable isotopically-coded biotin–azide that is installed using copper-catalyzed azide–alkyne chemistry. Following enrichment, the binding site is recovered and analyzed by isotope-targeted MS for high confidence detection and validation of the binding site structure. SIM-PAL enables identification of a broad range of molecular interactions, such as protein–protein interactions mediated by natural products. Rapamycin, an immunosuppressant macrocyclic natural product, forms a ternary complex with the proteins FKBP12 and the FRB domain of mTOR. SIM-PAL was applied to the FKBP12–rapamycin–FRB system to reveal multiple insights into photo-affinity labeling chemistry and the structural measurements it yields. Comparable protein binding activity with rapamycin and a photo-rapamycin analog was confirmed using surface plasmon resonance, indicating that the modified compound forms the desired ternary complex. In an in vitro system of purified FKBP and FRB, peptides modified by photo-rapamycin were identified by LC-MS/MS/MS using a combination of database searching and customized data analysis methods. The resulting binding site identifications reveal key characteristics of the interactions that are captured by photo-affinity labelling. SIM-PAL successfully mapped the rapamycin ternary complex in a manner analogous to previous in vitro structural characterization methods, implying the potential for this level of structural insight to be gained across a whole proteome with bioactive natural products.

Structural characterization of protein–small molecule interactions in ternary complexes using photo-affinity labelling and mass spectrometry: Studies with the macrocyclic natural product rapamycin

256th National Meeting (2018)

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