The SH2 domain-containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T-cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14-3-3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14-3-3/SLP76 protein-protein interaction (PPI), we have determined a high-resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14-3-3r. We then characterized its binding to 14-3-3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14-3-3. Our work lays the foundation for drug design efforts aimed at targeting the 14-3-3/SLP76 interaction and, thereby, TCR signalling.
14-3-3 proteins regulate many intracellular processes and their ability to bind in subtly different fashions to their numerous partner proteins provides attractive drug-targeting points for a range of diseases. Schnurri-3 is a suppressor of mouse bone formation and a candidate target for novel osteoporosis therapeutics, and thus it is of interest to determine whether it interacts with 14-3-3. In this work, potential 14-3-3 interaction sites on mammalian Schnurri-3 were identified by an in silico analysis of its protein sequence. Using fluorescence polarization, isothermal titration calorimetry and X-ray crystallography, it is shown that synthetic peptides containing either phosphorylated Thr869 or Ser542 can indeed interact with 14-3-3, with the latter capable of forming an interprotein disulfide bond with 14-3-3σ: a hitherto unreported phenomenon.
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