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  3. A biophysical and structural analysis of the interaction of BLNK with 14-3-3 proteins

A biophysical and structural analysis of the interaction of BLNK with 14-3-3 proteins

  • J Struct Biol. 2020 Dec 1;212(3):107662. doi: 10.1016/j.jsb.2020.107662.
Lorenzo Soini 1,  Seppe Leysen 2,  Jeremy Davis 3,  Christian Ottmann 4
Affiliations

Affiliations

  • 1 Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands; Department of Chemistry, UCB Celltech, Slough, UK.
  • 2 Department of Structural Biology and Biophysics, UCB Celltech, Slough, UK.
  • 3 Department of Chemistry, UCB Celltech, Slough, UK.
  • 4 Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands. Electronic address: c.ottmann@tue.nl.
PMID: 33176192 DOI: 10.1016/j.jsb.2020.107662
Abstract

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.

Keywords

Adaptor proteins 14-3-3 and BLNK; Gatekeeper; Isothermal titration calorimetry; Phosphorylation; X-ray protein crystallography.

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