Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA.

Abstract

The crystal structure of a ternary complex of human _nununodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p5i), a 19-base/18-base doublestranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. (40)(41)(42)(43)(44)(45). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and ringers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two a-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nudeophilic attack on the a-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies. A 3.5 A resolution structure of HIV-1 RT complexed with the nonnucleoside inhibitor nevirapine has been reported (8).Although the resolution of the study was not sufficient to determine the position of every amino acid and their side chains, the overall folding of the enzyme was described.We have prepared crystals of a ternary complex (9) of the HIV-1 RT p66/pSl heterodimer, a double-stranded DNA (dsDNA) template-primer, and the antigen-binding fragment (Fab fragment) ofa noninhibiting antibody that diffract x-rays to 2.8 A resolution, and reported the structure ofthis complex at 7 A resolution (10). At this resolution it was possible to determine the location ofthe template-primer and the relative positions of the polymerase and the RNase H active sites. In addition, it was shown that when the nucleic acid substrate was bound to RT a significant portion of the protein moved out of the nucleic acid-binding site.Here we report the structure of the RT-dsDNA-Fab28 complex at 3.0 A resolution. The overall arrangement of the enzyme is similar to that previously reported (8 ITo whom reprint requests should be addressed

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities

“…The numbers at the beginning of the sequences indicate the number of residues from the N-terminus of the RT-RH omitted from the Figure. minal nucleotide of the primer (the cleaved labelled RNA fragment has a length of 18 nt). In comparison, HIV-1 enzyme cleaved the RNA between positions 18 and 19, in keeping with biochemical and crystallographic data, indicating that, in HIV-1 RT, the distance between the positions of RH cleavage and DNA polymerase active sites is 18-20 bp [20][21][22][23][24][25][26][27]. The effect of primer extension on RH cleavage was examined ( Figure 5B, ' jdNTP ').…”

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

“…Two features enable us to build a feasible model using the EV71 RdRp apoenzyme structure: the direction of polymerase translocation along the template strand is consistent among the known polymerase structures; and the nascent nucleic acid duplex is known to adopt an approximate A-form conformation in the immediate vicinity of the active site (Jacobo-Molina et al, 1993;Doublie et al, 1998;Huang et al, 1998;Li et al, 1998;Lesburg et al, 1999). We therefore modeled the structure of EV71 RdRp (3D pol ) in complex with the template:primer (5'-CAUGGGCC-3'/5'-GGCCC-3') by superimposing the palm domain with the structure of the FMDV 3D polymerase in complex with the template:primer RNA (PDB code: 1WNE ) (Ferrer-Orta et al, 2004) (Fig.…”

Review

“…The RT-DNA interaction involves elements primarily from the thumb, fingers and palm subdomains of the p66 subunit. Other regions that are located near the template-primer include the connection subdomain and the RNase H domain of p66 (Jacobo-Molina et al, 1993;Ding et al, 1994). The monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer.…”

“…The double-stranded DNA bound to HIV-1 RT has an unusual and unexpected geometry. The majority of the 18 base pair duplex region is B-form, but in the vicinity of the polymerase active site there are about 6 to 7 base pairs of DNA which are in the A conformation (Jacobo-Molina et al, 1993). At the junction between the -form and B-form regions of the template-primer, there is a 40 to 45° bend that widens the minor groove.…”

“…These residues, which are well conserved within the pol genes of many retroviruses (motifs A and C, Larder et al, 1987b;Poch et al, 1989;Delarue et al, 1990), are involved in forming the topology of the nucleotide-binding site, which is located in the palm subdomain of the p66 subunit of HIV-1 RT. The most conserved element of this region in the HIV-1 RT and other lentiviruses have the YMDD motif (Tyr-Met-Asp-Asp amino acid residues 183-186) Jacobo-Molina et al, 1993). HIV-1 RTs in which these residues are mutated are inactive (Larder et al, 1989;Boyer, PL.…”