The 14‐3‐3/SLP76 protein–protein interaction in T‐cell receptor signalling: a structural and biophysical characterization

Abstract

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.

Identification of molecular glues of the SLP76/14-3-3 protein–protein interaction

“…The interaction between 14-3-3 proteins and SLP76 has been previously characterized exploiting a short synthetic phosphorylated peptide, SLP76pS376. 63 Fluorescence polarization binding assay identified 14-3-3γ as the isoform with highest affinity against such system. 63 Moreover, relative affinities of cellular SLP76 binding to 14-3-3 protein also report 14-3-3γ as the highest affinity isoform highlighting its biological importance.…”

“… 63 Fluorescence polarization binding assay identified 14-3-3γ as the isoform with highest affinity against such system. 63 Moreover, relative affinities of cellular SLP76 binding to 14-3-3 protein also report 14-3-3γ as the highest affinity isoform highlighting its biological importance. 27 14-3-3γ was therefore chosen as the isoform to perform the screening against.…”

“…A detailed characterisation of the 14-3-3γ/SLP76 PPI with the exact SLP76 construct used here has already been described in another work. 63 However, an additional SPR binding assay in the presence of 5% DMSO from which the K D is calculated is reported ( Fig. 1a ).…”

“…The expression, purification and phosphorylation of the SLP76 construct (SLP76-SH2) has been described before. 63 …”

“…SPR experiments were performed on a Biacore T200 (Cytiva). The Sensor Chip NTA was validated as previously reported by Soini et al 63 A DMSO tolerance at 2% and 5% was performed in the same way (Fig. S1 † ).…”