TY - JOUR T1 - Chimeric 14-3-3 proteins for unravelling interactions with intrinsically disordered partners JF - bioRxiv DO - 10.1101/130245 SP - 130245 AU - Nikolai N. Sluchanko AU - Kristina V. Tugaeva AU - Alfred A. Antson Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/04/24/130245.abstract N2 - In eukaryotes, several proteins act as “hubs”, integrating signals from a variety of interacting partners that bind to the hub through intrinsically disordered regions. Not surprisingly, one of the major hubs, the 14-3-3 protein, that plays wide-ranging roles in cellular processes, has been linked with a number of disorders including neurodegenerative diseases and cancer. A partner protein usually binds with its phosphopeptide accommodated in an amphipathic groove (AG) of 14-3-3, a promising platform for therapeutic intervention. Protein plasticity in the groove allows to accommodate a range of phosphopeptides with different sequences. So far, in spite of mammoth effort, accurate structural information has been derived only for few 14-3-3 complexes with phosphopeptide-containing proteins or various short synthetic peptides. The progress has been prevented by intrinsic disorder of partner proteins and, in case of transient interactions, by the low affinity of phosphopeptides. We reasoned that these problems could be resolved by using chimeric 14-3-3 proteins with incorporated peptide sequences. We tested this hypothesis and found that such chimeric proteins are easy to design, express, purify and crystallize. We show that when attached to the C terminus of 14-3-3 via an optimal linker, peptides become stoichiometrically phosphorylated by protein kinase A during bacterial co-expression. We determined crystal structures for complexes of chimeric 14-3-3 protein fused with three different peptides. In most of the cases, the phosphopeptide is bound inside the AG, providing invaluable information on its interaction with the protein. This approach can reinvigorate studies of 14-3-3 protein complexes, including those with otherwise challenging low affinity phosphopeptides. Furthermore, 14-3-3-phosphopeptide chimeras can be useful for the design of novel biosensors for in vitro and in vivo imaging experiments. ER -