Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.
We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.
Cheng
N
N
Division of Virology, Burroughs Wellcome Company, Research Triangle Park, North Carolina 27709.
Merrill
B M
BM
Painter
G R
GR
Frick
L W
LW
Furman
P A
PA
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010777:Photochemistry; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013942:Thymine Nucleotides
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D009711:Nucleotides; D013942:Thymine Nucleotides; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D008239:Lysine; C024157:thymidine 5'-triphosphate