title: Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.
abstract: HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners.
authors.affiliation: From the Departments of Microbiology and Molecular Genetics and Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and.
authors.forename: John Jeff
authors.initials: JJ
authors.lastname: Alvarado
authors.affiliation: From the Departments of Microbiology and Molecular Genetics and Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
authors.forename: Sreya
authors.initials: S
authors.lastname: Tarafdar
authors.affiliation: Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and jiyeh@pitt.edu.
authors.forename: Joanne I
authors.initials: JI
authors.lastname: Yeh
authors.affiliation: From the Departments of Microbiology and Molecular Genetics and tsmithga@pitt.edu.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008958:Models, Molecular; D010957:Plasmids / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D051572:Proto-Oncogene Proteins c-hck / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck
keywords: Hck Kinase; Human Immunodeficiency Virus (HIV); Src; Src Homology 2 Domain (SH2 Domain); Src Homology 3 Domain (SH3 Domain); X-ray Crystallography