Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label

Abstract

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.

Affinity modification of human immunodeficiency virus reverse transcriptase and DNA template by photoreactive dCTP analogs

“…The results speak in favour of the localisation of the dNTP binding site and the catalytic center of HIV-RT on the 66 kDa subunit of the heterodimer. These data are in agreement with direct dNTP UV-crosslinking to HIV-RT [8,9], photoaffinity labeling of the enzyme with rlthio UTP [lo], and X-ray analysis data obtained for HIV-RT at 3 A resolution [l 11. (1) enxyme incubated with templatsprimer hybrid, AlFABdCTP for 2.5 min and UV-irradiated for 2.5 min; (2) enzyme incubated for 1 h with template-primer hybrid and AlFABdCIP UV-irradiated before incubation; (3) enzyme irradiated with AlFABdCTP for 2.5 min followed by incubation for 1 h in the presence of template and primer; (4) enzyme irradiated with AlFABdCTP and template followed by addition of primer and incubation for 1 h; (5) enzyme incubated with template, primer and AlFABdCTP, for 2.5 min followed by UV-irradiation for 2.5 min.…”

Function and Structure Relationships in DNA Polymerases

“…Photo affinity labeling of a binary complex with dTTP resulted in crosslinking to Lys73 (78), and an antibody inhibition study suggested that dNTP binds in the vicinity of residues 65-73 (79). However, the residues implicated by these studies are located on the l3-hairpin that has been proposed, from crystallographic work, to bind to the template strand (Figure 9), and Lys73 is more than 15 A from the primer terminus (10, 11, 37; J Jager, TA Steitz, unpublished).…”

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV‐1 reverse transcriptase: tRNALys,3 activation

“…No crystallographic data is currently available of a HIV-1 RT. dNTP complex, but UV-induced crosslinking of labelled dTTP to HIV-1 RT has shown that within the p66 subunit, dTTP appears to cross-link to a single residue, Lys-73 [21]. Thus, the precursor site and the primer-template one are very close in the catalytic subunit of the viral polymerase.…”