title: Impact of H216 on the DNA binding and catalytic activities of the HIV restriction factor APOBEC3G.
abstract: APOBEC3G has an important role in human defense against retroviral pathogens, including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time-resolved nuclear magnetic resonance (NMR) spectroscopy to determine kinetic parameters of A3Gctd's deamination reactions within a 5'-CCC hot spot sequence. A3Gctd exhibited a 45-fold preference for 5'-CCC substrate over 5'-CCU substrate, which explains why A3G displays almost no processivity within a 5'-CCC motif. In addition, A3Gctd's shortest substrate sequence was found to be a pentanucleotide containing 5'-CCC flanked on both sides by a single nucleotide. A3Gctd as well as full-length A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo.
authors.affiliation: Department of Biochemistry, Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Harjes
authors.forename: William C
authors.initials: WC
authors.lastname: Solomon
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Kuan-Ming
authors.initials: KM
authors.lastname: Chen
authors.forename: Elena
authors.initials: E
authors.lastname: Harjes
authors.forename: Reuben S
authors.initials: RS
authors.lastname: Harris
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuo
mesh_terms: D000071480:APOBEC-3G Deaminase; D002384:Catalysis; D002460:Cell Line; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000472:pathogenicity*; D006639:Histidine / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D017434:Protein Structure, Tertiary
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D006639:Histidine; D000071480:APOBEC-3G Deaminase; C464924:APOBEC3G protein, human; D003564:Cytidine Deaminase