Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

• Published in: The Journal of biological chemistry 1998-12, Vol.273 (49), p.32685-32689
• Main Authors: Lubkowski, Jacek, Yang, Fan, Alexandratos, Jerry, Merkel, George, Katz, Richard A., Gravuer, Kelly, Skalka, Anna Marie, Wlodawer, Alexander
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Amino Acid Sequence; ANALYTICAL METHODS; Asparagine - chemistry; Aspartic Acid - chemistry; AVIAN ONCOVIRUS; Avian sarcoma virus; Avian Sarcoma Viruses - enzymology; BINDING; Binding Sites; Catalytic Domain; CATION; CATIONES; CATIONS; Cations, Divalent; CINC; CRYSTAL STRUCTURE; CRYSTALS; DIVALENT CATIONS; ENZIMAS; ENZYME; ENZYMES; Hydrogen Bonding; Hydrogen-Ion Concentration; Integrases - chemistry; Integrases - metabolism; MOLECULAR CONFORMATION; Molecular Sequence Data; MUTANT; MUTANTES; MUTANTS; Mutation; ONCOVIRUS AVIAIRE; ONCOVIRUS AVIAR; PDB/1VSK; PDB/1VSM; Protein Binding; Protein Conformation; TECHNIQUE ANALYTIQUE; TECNICAS ANALITICAS; X RAY DIFFRACTION; ZINC
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.49.32685

Crystallographic studies of the catalytic core domain of avian sarcoma virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs against AIDS. Recently, crystals of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations. Data were also collected at low pH for the crystals of the native ASV IN core domain. In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx-64 (Asx denotes Asn or Asp) is rotated by ∼150° around the Cα—Cβ bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn2+ can still bind to Asp-121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner.