Disentangling the Complexity in Protein Complexes Using Complementary Isotope-Labeling and Multiple-Receiver NMR Spectroscopy

Intrinsically disordered proteins are abundant in eukaryotic systems, but they remain largely elusive pharmacologicaltargets. NMR spectroscopy proved to be a suitable method to study these proteins and their interaction with one another or withdrug candidates. Although NMR can give atomistic information about these interplays, molecular complexity due to severe spectraloverlap, limited sample stability, and quantity remain an issue and hamper widespread applications. Here, we propose an approach tosimultaneously map protein−protein binding sites onto two interacting partners by employing a complementary isotope-labelingstrategy and a multiple receiver NMR detection scheme. With one partner being 15N,2H labeled and the interacting one being13C,1H-labeled, we exploited proton and carbon detection to obtain clean and easily readable information. The method is illustratedwith an application to the 50 kDa ternary protein complex formed between the prominent oncogenic transcription factor complexMyc/MAX and the tumor suppressor BRCA1