title: 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.
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 the V and 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.
authors.affiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian 350002, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Shi
authors.forename: Sandrine
authors.initials: S
authors.lastname: Opi
authors.forename: Adrien
authors.initials: A
authors.lastname: Lugari
authors.forename: Audrey
authors.initials: A
authors.lastname: Restouin
authors.forename: Thibault
authors.initials: T
authors.lastname: Coursindel
authors.forename: Isabelle
authors.initials: I
authors.lastname: Parrot
authors.forename: Javier
authors.initials: J
authors.lastname: Perez
authors.forename: Eric
authors.initials: E
authors.lastname: Madore
authors.forename: Pascale
authors.initials: P
authors.lastname: Zimmermann
authors.forename: Jacques
authors.initials: J
authors.lastname: Corbeil
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
authors.forename: Stefan T
authors.initials: ST
authors.lastname: Arold
authors.forename: Yves
authors.initials: Y
authors.lastname: Collette
authors.forename: Xavier
authors.initials: X
authors.lastname: Morelli
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D051572:Proto-Oncogene Proteins c-hck / Q000737:chemistry* / Q000378:metabolism; D053838:Scattering, Small Angle; D020798:Two-Hybrid System Techniques; D057074:Virus Release; D018909:src Homology Domains
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; D051572:Proto-Oncogene Proteins c-hck