Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase.

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.

Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance.

The locations of HIV-1 RT nucleoside and non-nucleoside inhibitor-binding sites and inhibitor-resistance mutations are analyzed in the context of the three-dimensional structure of the enzyme and implications for mechanisms of drug inhibition and resistance are discussed. In order to help identify residues that may play a role in inhibitor binding, solvent accessibilities of amino acids that comprise the inhibitor-binding sites in the structure of HIV-1 RT complexed with a dsDNA template-primer are analyzed. While some mutations that cause resistance to nucleoside analogs, such as AZT, ddI, and ddC, are located near enough to the dNTP-binding site to directly interfere with binding of nucleoside analogs, many are located away from the dNTP-binding site and more likely confer resistance by other mechanisms. Many of the latter mutations are located on the surface of the DNA-binding cleft and may lead to altered template-primer positioning or conformation, causing a distortion of the geometry of the polymerase active site and consequent discrimination between normal and altered dNTP substrates. Other nucleoside analog-resistance mutations located on the periphery of the dNTP-binding site may exert their effects via altered interactions with dNTP-binding site residues. The structure of the hydrophobic region in HIV-1 RT that binds non-nucleoside inhibitors, for example, nevirapine and TIBO, has been analyzed in the absence of bound ligand. The pocket that is present when non-nucleoside inhibitors are bound is not observed in the inhibitor-free structure of HIV-1 RT with dsDNA. In particular it is filled by Tyr181 and Tyr188, suggesting that the pocket is formed primarily by rotation of these large aromatic side-chains. Existing biochemical data, taken together with the three-dimensional structure of HIV-1 RT, makes it possible to propose potential mechanisms of inhibition by non-nucleoside inhibitors. One such mechanism is local distortion of HIV-1 RT structural elements thought to participate in catalysis: the beta 9-beta 10 hairpin (which contains polymerase active site residues) and the beta 12-beta 13 hairpin ("primer grip"). An alternative possibility is restricted mobility of the p66 thumb subdomain, which is supported by the observation that structural elements of the non-nucleoside inhibitor-binding pocket may act as a "hinge" for the thumb.(ABSTRACT TRUNCATED AT 400 WORDS)

Buried surface analysis of HIV-1 reverse transcriptase p66/p51 heterodimer and its interaction with dsDNA template/primer.

The p66/p51 human immunodeficiency virus type 1 reverse transcriptase is a heterodimer with identical N-terminal amino acid sequences. The enzyme contains two polymerization domains and one RNase H domain, which is located at the C-terminus of the p66 subunit. Both polymerization domains fold into four individual subdomains that are not arranged in a similar fashion, forming an unusually asymmetric dimer. The complexity of the RT p66/p51 heterodimer structure is simplified using solvent-accessibility surface areas to describe the buried surface area of contact among the different subdomains. In addition, the RT/DNA contacts in the recently published RT/DNA/Fab structure [Jacobo-Molina et al., Proc. Natl Acad. Sci. USA, 90, 6320-6324 (1993)] are described using the same approach. Finally, the RT/DNA complex is compared with other dimeric DNA-binding proteins. It was found that the size of the protein and the extent of the dimer interface were not directly related to the extent of contact between the protein and the DNA. Furthermore, RT, the only protein that is not a sequence-specific DNA binding protein in this analysis, had the largest surface of interaction with the nucleic acid.

Sensitivity of wild-type human immunodeficiency virus type 1 reverse transcriptase to dideoxynucleotides depends on template length; the sensitivity of drug-resistant mutants does not.

Analysis of the three-dimensional structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with double-stranded DNA indicates that while many nucleoside-resistance mutations are not at the putative dNTP binding site, several are in positions to interact with the template-primer. Wild-type HIV-1 RT and two nucleoside-resistant variants, Leu74-->Val and Glu89-->Gly, have been analyzed to determine the basis of resistance. The ability of the wild-type enzyme to incorporate, or reject, a 2',3'-dideoxynucleoside triphosphate (ddNTP) is strongly affected by interactions that take place between the enzyme and the extended template strand 3-6 nt beyond the polymerase active site. Inspection of a model of the enzyme with an extended template suggests that this interaction involves the fingers subdomain of the p66 subunit in the vicinity of Leu74. These data provide direct evidence that the fingers subdomain of the p66 subunit of HIV-1 RT interacts with the template strand. The wild-type enzyme is resistant to ddITP if the template extension is 3 nt or less and becomes sensitive only when the template extends more than 3 or 4 nt beyond the end of the primer strand. However, the mutant enzymes are resistant with both short and long template extensions. Taken together with the three-dimensional structure of HIV-1 RT in complex with double-stranded DNA, these data suggest that resistance to the dideoxynucleotide inhibitors results from a repositioning or change in the conformation of the template-primer that alters the ability of the enzyme to select or reject an incoming dNTP.

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

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). 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 fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-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 nucleophilic attack on the alpha-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.

Structure determination of antiviral compound SCH 38057 complexed with human rhinovirus 14.

SCH 38057 (1-[6-(2-chloro-4-methoxyphenoxy)-hexyl]imidazole hydrochloride) is a new, water-soluble antiviral compound that has inhibitory activities against a number of picornavirus infections. The structure of the human rhinovirus 14 (HRV14) complex with SCH 38057 was determined at 3.0 A resolution by single-crystal diffraction techniques using synchrotron X-radiation. SCH 38057 was found to bind at the innermost end of the hydrophobic pocket within the capsid protein VP1, a locus of binding of other antipicornaviral agents; however, the complex differs from previously reported complexes in two important aspects. It leaves a considerable volume near the entrance to the binding pocket unoccupied. In addition, the alterations in the conformation of the VP1 polypeptide are similar to, but more extensive than those observed in HRV14 complexes with other antiviral agents. Although only 9 amino acids of VP1 have close contacts with the SCH 38057 molecule (within 3.6 A), at least 36 amino acids from both VP1 and VP3 have significantly altered conformations (C alpha movement > 0.5 A versus native). The structures of complexes of HRV14 with SCH 38057 and WIN 51711 are compared. Aromatic ring interactions between picornavirus capsid residues and antiviral inhibitors are proposed to be among the major determinants for positioning of these compounds.

Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations.

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.

Crystals of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase with a monoclonal antibody Fab fragment and double-stranded DNA diffract x-rays to 3.5-A resolution.

Two crystal forms of complexes have been grown that contain human immunodeficiency virus type 1 reverse transcriptase and a monoclonal antibody Fab fragment. One of the crystal forms (form II, space group P3112, a = 168.7 A, c = 220.3 A) diffracts x-rays to 3.5-A resolution and appears suitable for moderate-resolution structure determination. The form II crystals have the unusual property that their maximum resolution of diffraction and resistance to radiation damage are enhanced by either crystallization in the presence of or soaking with double-stranded DNA primer-template mimics. These crystals may permit structural studies of catalytically relevant complexes and eventually enable us to experimentally observe successive steps in the reverse transcription process.