title: Structure, dynamics, and Hck interaction of full-length HIV-1 Nef.
abstract: Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72-256; Hck32L). A helical conformation was found at the N-terminus for residues 14-22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1-39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated, full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with (15)N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.
authors.affiliation: Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.
authors.forename: Jinwon
authors.initials: J
authors.lastname: Jung
authors.forename: In-Ja L
authors.initials: IJ
authors.lastname: Byeon
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Ahn
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
mesh_terms: D015658:HIV Infections / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D051572:Proto-Oncogene Proteins c-hck / Q000737:chemistry / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D054311:nef Gene Products, Human Immunodeficiency Virus; D051572:Proto-Oncogene Proteins c-hck