TY - JOUR
T1 - A biophysical and structural analysis of the interaction of BLNK with 14-3-3 proteins
AU - Soini, Lorenzo
AU - Leysen, Seppe
AU - Davis, Jeremy
AU - Ottmann, Christian
PY - 2020/12/1
Y1 - 2020/12/1
N2 - B-cell linker protein (BLNK) is an adaptor protein that orchestrates signalling downstream of B-cell receptors. It has been reported to undergo proteasomal degradation upon binding to 14-3-3 proteins. Here, we report the first biophysical and structural study of this protein-protein interaction (PPI). Specifically, we investigated the binding of mono- and di- phosphorylated BLNK peptides to 14-3-3 using fluorescent polarization (FP) and isothermal titration calorimetry assays (ITC). Our results suggest that BLNK interacts with 14-3-3 according to the gatekeeper model, where HPK1 mediated phosphorylation of Thr152 (pT152) allows BLNK anchoring to 14-3-3, and an additional phosphorylation of Ser285 (pS285) by AKT, then further improves the affinity. Finally, we have also solved a crystal structure of the BLNKpT152 peptide bound to 14-3-3σ. These findings could serve as important tool for compound discovery programs aiming to modulate this interaction with 14-3-3.
AB - B-cell linker protein (BLNK) is an adaptor protein that orchestrates signalling downstream of B-cell receptors. It has been reported to undergo proteasomal degradation upon binding to 14-3-3 proteins. Here, we report the first biophysical and structural study of this protein-protein interaction (PPI). Specifically, we investigated the binding of mono- and di- phosphorylated BLNK peptides to 14-3-3 using fluorescent polarization (FP) and isothermal titration calorimetry assays (ITC). Our results suggest that BLNK interacts with 14-3-3 according to the gatekeeper model, where HPK1 mediated phosphorylation of Thr152 (pT152) allows BLNK anchoring to 14-3-3, and an additional phosphorylation of Ser285 (pS285) by AKT, then further improves the affinity. Finally, we have also solved a crystal structure of the BLNKpT152 peptide bound to 14-3-3σ. These findings could serve as important tool for compound discovery programs aiming to modulate this interaction with 14-3-3.
KW - Adaptor proteins 14-3-3 and BLNK
KW - Gatekeeper
KW - Isothermal titration calorimetry
KW - Phosphorylation
KW - X-ray protein crystallography
UR - http://www.scopus.com/inward/record.url?scp=85095934672&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2020.107662
DO - 10.1016/j.jsb.2020.107662
M3 - Article
C2 - 33176192
AN - SCOPUS:85095934672
SN - 1047-8477
VL - 212
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
M1 - 107662
ER -