Abstract
The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence. A stable ternary elongation complex was assembled containing enzyme, DNA template (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodideoxyuridine triphosphate was incorporated uniquely at the 3' terminus of the 32P-labeled DNA product. Upon photolysis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either trypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separated, purified, and finally subjected to Edman microsequencing. A unique radioactive hexapeptide (V276RQLCK281) was identified and sequenced. Our photoaffinity labeling results were positioned on the HIV-1 RT. DNA.Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site.
Highlights
The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence
The enzyme reverse transcriptase (RT)1 derived from the HIV-1 virus is a heterodimer composed of two subunits p66 and p51, which are derived from the same sequence
The results reported in this paper utilized the photoaffinity probe S4-ddUTP to derivatize the active site of HIV-1 RT during productive synthesis involving a ternary complex
Summary
(Received for publication, July 24, 1997, and in revised form, October 10, 1997). From the ‡Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 and ¶Genentech, South San Francisco, California 94080. Our photoaffinity labeling results were positioned on the HIV-1 RT1⁄7DNA1⁄7Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site. Studies involving specific amino acid derivatizations have suggested that Lys263 [10] and Arg277 [11] are critically involved at the active site. Photoaffinity labeling studies have yielded additional suggestions for the nucleotide binding site components: Lys73 [12], residues 288 –307 [13], and residues 288 – 423 [14]. Model building efforts based primarily on the RT1⁄7dsDNA1⁄7Fab x-ray structure have yielded a detailed mechanistic proposal for the HIV-1 RT enzyme [19]. The results reported in this paper utilized the photoaffinity probe S4-ddUTP to derivatize the active site of HIV-1 RT during productive synthesis involving a ternary complex. A general discussion of the possible mechanistic implications of these results is directed at making all the known topologic information compatible
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