title: Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor.
abstract: The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wales
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: James M
authors.initials: JM
authors.lastname: Hochrein
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Morgan
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Emert-Sedlak
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15219, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
authors.affiliation: Department of Chemistry & Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
mesh_terms: D004789:Enzyme Activation / Q000187:drug effects; D015658:HIV Infections / Q000188:drug therapy* / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation / Q000187:drug effects; D060066:Protein Interaction Maps / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051572:Proto-Oncogene Proteins c-hck / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D047428:Protein Kinase Inhibitors; D054311:nef Gene Products, Human Immunodeficiency Virus; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck