Structure of HIV-1 reverse transcriptase in a complex with the non-nucleoside inhibitor alpha-APA R 95845 at 2.8 A resolution.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that copies the RNA genome of HIV-1 into DNA. It is a heterodimer composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. HIV-1 RT is a crucial target for structure-based drug design, and potent inhibitors have been identified, whose efficacy, however, is limited by drug resistance. RESULTS: The crystal structure of HIV-1 RT in complex with the non-nucleoside inhibitor alpha-anilinophenyl-acetamide (alpha-APA) R95845 has been determined at 2.8 A resolution. The inhibitor binds in a hydrophobic pocket near the polymerase active site. The pocket contains five aromatic amino acid residues and the interactions of the side chains of these residues with the aromatic rings of non-nucleoside inhibitors appear to be important for inhibitor binding. Most of the amino acid residues where mutations have been correlated with high levels of resistance to non-nucleoside inhibitors of HIV-1 RT are located close to alpha-APA. The overall fold of HIV-1 RT in complex with alpha-APA is similar to that found when in complex with nevirapine, another non-nucleoside inhibitor, but there are significant conformational changes relative to an HIV-1 RT/DNA/Fab complex. CONCLUSIONS: The non-nucleoside inhibitor-binding pocket has a flexible structure whose mobility may be required for effective polymerization, and may be part of a hinge that permits relative movements of two subdomains of the p66 subunit denoted the 'palm' and 'thumb'. An understanding of the structure of the inhibitor-binding pocket, of the interactions between HIV-1 RT and alpha-APA, and of the locations of mutations that confer resistance to inhibitors provides a basis for structure-based design of chemotherapeutic agents for the treatment of AIDS.

Crystallization and preliminary X-ray diffraction analysis of oucleoside diphosphate kinase from Myxococcus xanthus.

Nucleoside diphosphate (NDP) kinase catalyzes the transfer of the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. Human and rodent forms of this enzyme have been shown to be suppressors of metastasis. Crystals that diffract X-rays to high resolution have been obtained for the recombinant Myxococcus xanthus NDP kinase expressed in and purified from Escherichia coli. Two crystal forms have been obtained. Both forms are orthorhombic, space group I222 (or I2(1)2(1)2(1)) with a = 267.1 A, b = 74.0 A and c = 75.1 A for form I and a = 53.5 A, b = 74.0 A and c = 75.1 A for form II. Form I appears to have five molecules in the asymmetric unit approximately related to each other by a translation of 0.2 along the a axis. Diffraction data have been recorded to 1.9 A for form I and to 2.2 A for form II.

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)

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 and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 A resolution.

The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the template-primer (including the primer grip, template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.

Proteolytic signal sequences (PEST) in the mammalian aminoacyl-tRNA synthetase complex.

Eight aminoacyl-tRNA synthetases together with three unidentified proteins are associated as a multi-enzyme complex in mammalian cells. Partial peptide sequences for lysyl- and aspartyl-tRNA synthetases are determined and no highly hydrophobic peptides are found. The partial amino acid sequences for two of the unidentified proteins in the complex are shown to have substantial homology and each has a number of unique sequences. The results suggest that the two unidentified proteins are fragments of synthetases. The partial sequences revealed the presence of PEST sequences in at least three proteins. Inasmuch as PEST sequences are signals for intracellular degradation, the mammalian synthetase complex may have evolved to protect these synthetases against intracellular proteolysis.

HIV reverse transcriptase structure-function relationships.

HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented.

Major subdomain rearrangement in HIV-1 reverse transcriptase simulated by molecular dynamics.

We have performed eight 1-ns in vacuo molecular dynamics simulations of HIV-1 reverse transcriptase (RT). Starting with the p66 thumb subdomain in an upright configuration, the p66 thumb moved down over the palm during six of the eight trajectories, in excellent agreement with the crystallographic structure of unliganded RT. The large rearrangement of the p66 thumb subdomain, its tip moving approximately 30 A, occurs during the first 30-200 ps. This approach may allow a detailed study of the processes involved in biologically significant conformational changes in macromolecules.

cDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase.

Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes. Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery. To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase. The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase. The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus. The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics. Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase. The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.

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.

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.

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.

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.

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.

Design and construction of rhinovirus chimeras incorporating immunogens from polio, influenza, and human immunodeficiency viruses.

This paper describes the design and construction of chimeric human rhinoviruses that contain immunogenic regions from other pathogens as part of their surface coat proteins. Segments encoding the poliovirus 3 Sabin VP1 and VP2 proteins, the influenza hemagglutinin (HA) glycoprotein, and the human immunodeficiency virus gp120 surface and gp41 transmembrane glycoproteins were inserted into a full-length clone of human rhinovirus 14 (HRV14) at regions corresponding to neutralizing immunogenic sites IA (NIm-IA) and II (NIm-II). Of 12 chimeric constructs described, 3 produced viable virus. An HRV14 chimeric virus containing five amino acids of influenza HA (corresponding to 300 A2 of solvent-accessible surface area) had wild-type HRV14 growth characteristics and was neutralized by four of four anti-influenza HA antisera with reciprocal neutralizing titers ranging from 180 to 330. However, antisera raised in two guinea pigs against the HRV14:influenza HA chimera did not show significant neutralization of relevant strains of influenza. These results are the first to demonstrate the feasibility of making viable chimeras of human rhinoviruses displaying heterologous immunogens.