Title: Identification and biophysical assessment of the molecular recognition mechanisms between the human hemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X
Authors: Shi, Xiaoli
Opi, Sandrine
Lugari, Adrien
Restouin, Audrey
Coursindel, Thibault
Parrot, Isabelle
Perez, Javier
Madore, Eric
Zimmermann, Pascale
Corbeil, Jacques
Huang, Mingdong
Arold, Stefan T
Collette, Yves
Morelli, Xavier # ×
Issue Date: Oct-2010
Publisher: Published by Portland Press on behalf of the Biochemical Society
Series Title: Biochemical Journal vol:431 pages:93-102
Abstract: Src family kinases (SFKs) are central regulators of many signaling 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 (hemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione S-transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cellulo. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 with a dissociation constant of 34.5 microM. Heteronuclear NMR spectroscopy experiments was used to map Hck-SH3 residues that interact with the ALIXV+PRR construct or with the minimum identified interacting motif. Finally, small-angle X-ray scattering (SAXS) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. Our results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity toward Hck-SH3. The structural framework of the Hck-Alix interaction demonstrated here will help in clarifying how Hck and Alix assist during virus budding and cell surface receptor regulation.
ISSN: 0264-6021
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Department of Human Genetics - miscellaneous
× corresponding author
# (joint) last author

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