Identification and biophysical assessment of the molecular recognition mechanisms between the human haemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X

Xiaoli Shi, Sandrine Opi, Adrien Lugari, Audrey Restouin, Thibault Coursindel, Isabelle Parrot, Javier Perez, Eric Madore, Pascale Zimmermann, Jacques Corbeil, Mingdong Huang, Stefan Arold, Yves Collette, Xavier Morelli*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein-protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 μM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing theVand PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.

Original languageEnglish (US)
Pages (from-to)93-102
Number of pages10
JournalBiochemical Journal
Volume431
Issue number1
DOIs
StatePublished - Oct 1 2010

Keywords

  • Apoptosis-linked gene 2-interacting protein X (Alix)
  • Haemopoietic cell kinase (Hck)
  • Protein-protein interaction
  • Src family kinase (SFK)
  • Src homology 3 domain (SH3 domain)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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