Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV- 1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide

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 α-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodide-oxyuridine 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 (V276RQLCK261) 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.