Cross-linking of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase to template-primer.

Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used--photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N(2)-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5' extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the "open" configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.

Application of 3-[3-(3-(trifluoromethyl)diazirin-3-yl)phenyl]-2,3- dihydroxypropionic acid, carbene-generating, cleavable cross-linking reagent for photoaffinity labeling.

N-Hydroxysuccinimide ester of 3-[3-(3-(trifluoromethyl)diazirin-3-yl)phenyl]-2,3-dihydroxypro pionic acid was successfully tested in a ribosomal tRNA binding system. It is an originally designed trifluoromethyl-diazirine-based cleavable cross-linking reagent with a very short distance between the active points (about 8.5 A). The reagent was coupled to the amino acid amino group of Phe-tRNAPhe to obtain a photoactivatable analog of peptidyl-tRNA. This analog was bound to ribosomes and the complex was irradiated with uv light. After isolation, the cross-linked product was cleft by periodate treatment to reveal the properties of the new reagent.