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
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 # ×
Published by Portland Press on behalf of the Biochemical Society
Biochemical Journal vol:431 pages:93-102
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.