Identification of Amino Acids in HIV-1 and Avian Sarcoma Virus Integrase Subsites Required for Specific Recognition of the Long Terminal Repeat Ends
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m510628200
ISSN1083-351X
AutoresAiping Chen, Irene T. Weber, Robert W. Harrison, Jonathan Leis,
Tópico(s)RNA and protein synthesis mechanisms
ResumoA tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ′ process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val72, Ser153, Lys160–Ile161, Gly163–Val165, and His171–Leu172. Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser153 and Val72 in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance. A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ′ process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val72, Ser153, Lys160–Ile161, Gly163–Val165, and His171–Leu172. Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser153 and Val72 in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance. HIV-1 2The abbreviations used are: HIV, human immunodeficiency virus; IN, integrase; LTR, long terminal repeat; MuLV, murine leukemia virus; ASV, avian sarcoma virus; RSV, Rous sarcoma virus; SIV, simian sarcoma virus; oligo, oligodeoxyribonucleotide; PDB, Protein Data Bank; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid. DNA integration is a concerted process that occurs in defined stages. After assembly of a stable complex of the viral integrase (IN) and host cell proteins with specific DNA sequences at the end of the HIV-1 LTR, the terminal dinucleotides are removed from each 3′ end by endonucleolytic processing. The viral DNA 3′ ends are then covalently linked to the host cell target DNA in a concerted reaction. Biochemical details concerning the processing and joining steps in the integration process have been reported for several retroviruses including avian sarcomaleukosis virus (ASV), murine leukemia virus (MuLV), and HIV-1. Specific DNA sequences at the 3′ end of the viral LTR are required for recognition by the assembled viral integrase complex. Typically, the terminal 12–20 nucleotides are sufficient. After 3′ processing of the CAXX sequence from the ends, viral DNA ends are joined by the viral integrase and the gapped intermediates are repaired, and unpaired viral 5′ ends are excised by host nucleases. This results in a 4–6-base pair duplication of host cell DNA, depending upon the virus, flanking the integrated viral DNA. Both the 3′ processing and viral DNA-host DNA joining steps require integrase to be assembled on the specific viral DNA substrate. Integration of viral into host DNA occurs with limited sequence specificity (1.Kulkosky J. Skalka A.M. Pharmacol. Ther. 1994; 61: 185-203Crossref PubMed Scopus (52) Google Scholar). Several cell proteins have been reported to affect the integration process (2.Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar, 3.Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 4.Hindmarsh P. Ridky T. Reeves R. Andrake M. Skalka A.M. Leis J. J. Virol. 1999; 73: 2994-3003Crossref PubMed Google Scholar, 5.Chen H. Engelman A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15270-15274Crossref PubMed Scopus (168) Google Scholar, 6.Harris D. Engelman A. J. Biol. Chem. 2000; 275: 39671-39677Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 7.Kalpana G. Reicin A. Cheng G. Sorin M. Paik S. Goff S. Virology. 1999; 259: 274-285Crossref PubMed Scopus (29) Google Scholar, 8.Suzuki Y. Craigie R. J. Virol. 2002; 76: 12376-12380Crossref PubMed Scopus (60) Google Scholar). Much of the information available concerning the molecular mechanism of integration comes from the use of reconstituted systems employing duplex oligodeoxyribonucleotides (oligos). The ASV IN, for example, catalyzes specific cleavage at the 3′ end of the strands adjacent to the conserved CA dinucleotide using 15-bp substrates corresponding to the ASV U3 or U5 termini (9.Katzman M. Katz R.A. Skalka A.M. Leis J. J. Virol. 1989; 63: 5319-5327Crossref PubMed Google Scholar, 10.Kukolj G. Skalka A.M. Genes Dev. 1995; 20: 2556-2567Crossref Scopus (38) Google Scholar). Similar substrates were used to demonstrate the joining reaction in which one oligo integrated into another (11.Craigie R. Fujiwara T. Bushman F. Cell. 1990; 62: 829-837Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 12.Katz R.A. Merkel G. Kulkosky J. Leis J. Skalka A.M. Cell. 1990; 63: 87-95Abstract Full Text PDF PubMed Scopus (297) Google Scholar). For HIV-1 duplex oligo substrates of comparable size, selected nucleotide substitutions in the U5 and U3 LTR regions were shown to affect one or both of the catalytic functions of HIV-1 integrase. Nucleic acid substitutions in the HIV-1 U5 LTR region, for example, can inhibit 3′ processing (e.g. positions 1–5 and 9–11) or not (e.g. positions 6–8 and 12–14) (13.Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (263) Google Scholar). Other changes at specific nucleic acid positions in the HIV-1 U3 and U5 LTR regions can affect each of the catalytic reactions, although changes in one region can be more pronounced than in the other. Single LTR end duplex oligos are valuable tools for examining IN catalytic activity. Nevertheless, most do not display the concerted nature of the DNA integration reaction that was first demonstrated by Goff and co-worker (14.Murphy J.E. Goff S.P. J. Virol. 1992; 66: 5092-5095Crossref PubMed Google Scholar). In these experiments, deletions were introduced 5′ to the conserved CA dinucleotide in the MuLV U3 LTR region. When cells were infected with the mutant viruses, processing at the ends of both the U3 and U5 LTR regions was affected adversely. This finding implies that the two ends of the viral DNA were brought together at the insertion site such that mutations in one affected the processing of the other. A homodimer of IN supports 3′ processing and one-ended joining, whereas a homotetramer of IN supports these activities in a two-ended concerted DNA integration reaction (15.Faure A. Calmels C. Desjobert C. Castroviejo M. Caumont-Sarcos A. Tarrago-Litvak L. Litvak S. Parissi V. Nucleic Acids Res. 2005; 33: 977-986Crossref PubMed Scopus (167) Google Scholar). Thus the biologically important form of IN is likely to be a tetramer. Several assay systems that display concerted DNA integration properties have been described and used to demonstrate that changes in retroviral DNA integration are context-dependent. For example, base pair substitutions in the dominant LTR (HIV-1 U5 and ASV U3) cause significant decreases in the rate of catalysis. In contrast, comparable substitutions in the nondominant LTR, the HIV-1 U3 and ASV U5 LTR regions, are associated with changes in the mechanism from concerted to nonconcerted DNA integration (2.Aiyar A. Hindmarsh P. Skalka A.M. Leis J. J. Virol. 1996; 70: 3571-3580Crossref PubMed Google Scholar, 16.Brin E. Leis J. J. Biol. Chem. 2002; 277: 10938-10948Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 17.Brin E. Leis J. J. Biol. Chem. 2002; 277: 18357-18364Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 18.Fitzgerald M.L. Vora A.C. Zeh W.G. Grandgenett D.P. J. Virol. 1992; 66: 6257-6263Crossref PubMed Google Scholar, 19.Hindmarsh P. 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HIV-1 integrase is a 288-amino acid protein composed of three functional domains that are required for each of the catalytic reactions (25.Engelman A. Englund G. Orenstein J. Martin M. Craigie R. J. Virol. 1995; 69: 2729-2736Crossref PubMed Scopus (0) Google Scholar). The conserved N-terminal domain (residues 1–50) contains a HHCC zinc-binding site, and binding of Zn2+ to this domain may participate in promoting the formation of IN oligomers (26.Lee S.P. Han M.K. Biochemistry. 1996; 35: 3837-3844Crossref PubMed Scopus (68) Google Scholar, 27.Lee S.P. Xiao J. Knutson J.R. Lewis M.S. Han M.K. Biochemistry. 1997; 36: 173-180Crossref PubMed Scopus (157) Google Scholar, 28.Zheng R. Jenkins T.M. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13659-13664Crossref PubMed Scopus (309) Google Scholar, 29.Ellison V. Gerton J. Vincent K.A. Brown P.O. J. Biol. Chem. 1995; 270: 3320-3326Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The C-terminal domain, the least conserved of the three domains in terms of amino acid sequence, is composed of a bundle of three α-helices that form a Src homology-3 fold. This domain has nonspecific DNA binding activity and determinants for multimerization (30.Andrake M. Skalka A.M. J. Biol. Chem. 1996; 271: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The catalytic core domain contains residues required for catalytic activity, including a conserved DDE motif that binds the required metal cofactor (1.Kulkosky J. Skalka A.M. Pharmacol. Ther. 1994; 61: 185-203Crossref PubMed Scopus (52) Google Scholar). Photo-cross-linking reagents have defined areas of the HIV-1 IN surface in close proximity to the LTR ends (13.Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Crossref PubMed Scopus (263) Google Scholar, 31.Heuer T.S. Brown P.O. Biochemistry. 1997; 36: 10655-10665Crossref PubMed Scopus (134) Google Scholar, 32.Heuer T.S. Brown P.O. Biochemistry. 1998; 37: 6667-6678Crossref PubMed Scopus (155) Google Scholar, 33.Jenkins T.M. Esposito D. Engelman A. Craigie R. EMBO J. 1997; 16: 6849-6859Crossref PubMed Scopus (220) Google Scholar, 34.Drake R.R. Meamati N. Hong H. Pilon A.A. Sunthankar P. Hume S.D. Milne G.W.A. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4170-4175Crossref PubMed Scopus (61) Google Scholar). The viral DNA sequences stimulate high affinity binding of drugs that target the catalytic functions of integrase, suggesting that integrase may assume a distinct enzymatically active conformation following assembly (35.Pommier Y. Johnson A.A. Marchand C. Nature. 2005; 4: 236-248Google Scholar, 36.Asante-Appiah E. Skalka A.M. Adv. Virus Res. 1999; 52: 351-369Crossref PubMed Google Scholar). No full-length integrase structure has yet been determined by x-ray crystallography. Therefore, little is known about how the three domains are positioned relative to one another in the active oligomeric state (37.Craigie R. J. Biol. Chem. 2001; 276: 23213-23216Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 38.Petit C. Schwartz O. Mammano F. J. Virol. 1999; 73: 5079-5088Crossref PubMed Google Scholar) or what specific amino acid residues are involved in recognition of the viral DNA substrate. Some insight into the assembly of a stable complex of integrase with specific DNA sequences can be gleaned from DNA transposable elements. Such elements share a common mechanism of integration with retroviruses, and the catalytic domains of ASV and HIV integrases are highly homologous to those of Mu, Tn5, and Tc3 transposases (39.Davies D. Goryshin I. Reznikoff W. Rayment I. Science. 2000; 289: 77-85Crossref PubMed Scopus (321) Google Scholar, 40.Mizuuchi K. Annu. Rev. Biochem. 1992; 61: 1011-1051Crossref PubMed Scopus (314) Google Scholar, 41.Mizuuchi K. Genes Cells. 1997; 2: 1-12Crossref PubMed Scopus (79) Google Scholar, 42.Graig N.L. Science. 1995; 270: 253-254Crossref PubMed Scopus (150) Google Scholar, 43.Douglas R.D. Lisa M.B. William S.R. Ivan R. J. Biol. Chem. 1999; 274: 11904-11913Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The Tc3 transposase is a 329-amino acid protein with an N-terminal DNA binding domain, a discreet second DNA binding domain flanking the N-terminal domain, and a catalytic core domain with a DDE motif (44.van Pouderoyen G. Ketting R.F. Perrakis A. Plasterk R.H. Sixma T.K. EMBO. 1997; 16: 6044-6054Crossref PubMed Scopus (91) Google Scholar). For the following study, we developed a structural model for HIV-1 IN with bound LTR DNA ends. This model was used to predict amino acid interactions between residues on the IN surface and the LTRs. By substituting amino acids from ASV IN into the structurally related positions of HIV-1, we identified a series of residues that participate in the specific recognition of the LTR ends. Reagents—[γ-33P]ATP (2500 Ci/mmol), HiTrap™ chelating HP resin, and HiTrap™ heparin HP resin were purchased from Amersham Biosciences. T4 polynucleotide kinase was from United States Biochemical (Cleveland, OH). Isopropyl β-d-thiogalactopyranoside was from Roche Applied Science. A Slide-A-Lyzer dialysis cassette was obtained from Pierce. Centriprep centrifugal filter devices with YM-10 MW membranes were from Millipore (Bedford, MA). Acrylamide and bisacrylamide solutions were from Bio-Rad. Simple Blue Safestain was from Invitrogen. DE81 filters were purchased from Whatman. Unless specified, all restriction enzymes were purchased from New England Biolabs (Beverly, MA). ASV IN was provided by Dr. Ann Skalka (Fox Chase Cancer Center, Philadelphia, PA). Bacterial Strains and Growth Conditions—The protein expression host cells, BL21 (DE3), were purchased from Novagen (Madison, WI). An expression construct for HIV-1 IN 1–288 residues (p28bIN-3CS-F185H) was obtained from the laboratory of Dr. Ann Skalka and was constructed by Dr. Mark Andrake. The construct contains the wild type NY5 HIV-1 sequence of the IN gene (Parke-Davis clone) from the NdeI to the HindIII site in the pET28b plasmid vector. The IN sequence encodes four substitutions (C56S, C65S, C280S, and F185H) to increase the solubility of HIV-1 IN and a six-amino acid His tag and thrombin cleavage site at the N terminus of the gene. A translation stop codon was added after residue Asp288. Preparation of Duplex Oligo Substrates—The following oligodeoxyribonucleotides were used in the integrase activity assay shown in Scheme 1. The plus-strand substrates (100 pmol, containing the conserved "CA" dinucleotides) were 5′ end-labeled using T4 polynucleotide kinase (30 U) and 2 μl of [γ-33P]ATP as described previously (45.Johnson M. Morris S. Chen A. Stavnezer E. Leis J. BMC Biol. 2004. 2004; 2/8Google Scholar). The specific activity of the radiolabeled substrates was diluted to 105 cpm/pmol using an unlabeled processed strand oligo, and the mixture was purified and recovered from a 20% denaturing polyacrylamide gel. Duplex oligos were formed by annealing to a molar excess of unlabeled complementary strand as described (46.Jenkins T.M. Engelman A. Ghirlando R. Craigie R. J. Biol. Chem. 1996; 271: 7712-7718Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Construction of Chimera HIV-1/ASV INs—Mutagenesis oligodeoxyribonucleotides were obtained from Integrated DNA Technologies Inc. (Coralville, IA) and are listed in the table found in the supplemental data. The mutations were constructed using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to manufacturer's directions. Codon preferences for Escherichia coli were used in the design of the oligos. For chimeras S54–57, the mutations were introduced by the PCR overlap extension method (47.Aiyar A. Leis J. BioFeedback. 1993; 14: 366-367Google Scholar). The presence of all mutations was confirmed by sequencing the complete individual DNA clones. A gel extraction kit (Qiagen, Valencia, CA) was used to purify all PCR products. The Minipreps DNA purification system (Promega, Madison, WI) was used to prepare DNAs. Purification of HIV-1 IN and Chimeras—His-tagged HIV-1 IN and chimeras were purified as described previously (48.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar) with some modification. Briefly, expression of proteins was induced in BL21 (DE3) cells at 20 °C by adding isopropyl β-d-thiogalactopyranoside to 0.5 mm after the bacteria had grown to optical density at an A600 of 0.8. Bacteria were lysed in 25 mm bis-Tris, pH 6.1, 1 m KCl, 1 m urea, 1% thiodiglycol, and 5 mm imidazole and then filtered through an 0.2-μm membrane from Nalgene (Rochester, NY). The lysates fraction was applied to a HiTrap™ chelating HP nickel-affinity column (5 ml) (Amersham Biosciences), and IN was eluted with a 5 mm to 1.0 m linear imidazole gradient. Fractions containing partially purified IN, detected by absorbency at 280 nm, were pooled and applied to a HiTrap™ heparin HP column (5 ml), and His-tagged IN was eluted with a 0.25–1.0 m linear KCl gradient. The pooled heparin IN fractions were concentrated using a Centriprep filter with YM-10 MW membrane and then dialyzed against 25 mm bis-Tris, pH 6.1, 0.5 m KCl, 1% thiodiglycol, 1 mm dithiothreitol, 0.1 mm EDTA, 40% glycerol. The purified protein was aliquoted and stored at –80 °C. The protein concentration was determined using a Bio-Rad protein assay as described by the manufacturer. The N-terminal poly His tag was removed from S160–161 and S153 chimeras using a thrombin kit from Novagen as described by the manufacturer. Briefly, the cleavage reaction in 50 μl contained cleavage buffer, 10 μg of IN, and 0.1 unit of thrombin (diluted 1:10) and was incubated at room temperature for 30 min. An additional 0.1 unit of thrombin was then added, and the reaction was incubated for an additional 30 min. Twenty μl of a 50% slurry of nickel-nitrilotriacetic acid resins (Novage) equilibrated with 25 mm bis-Tris, pH 6.1, was added to the reaction mixture and stirred gently for 30 min at 4 °C. The resin was collected by centrifugation at 15,000 × g for 1 min. The supernatant containing the thrombin was discarded. The resin was washed with 25 mm bis-Tris buffer, pH 6.1, and then the processed IN was eluted with 150 mm imidazole buffer. Integrase 3′ End Processing Assay Using Duplex Oligodeoxyribonucleotide Substrates—The processing reactions for the HIV-1 U5 LTR substrate were carried out as described previously (46.Jenkins T.M. Engelman A. Ghirlando R. Craigie R. J. Biol. Chem. 1996; 271: 7712-7718Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Reactions were in a volume of 10 μl with 25 mm MOPS, pH 7.2, 10 mm dithiothreitol, 15 mm potassium glutamate, 5% polyethylene glycol 8000, 5% Me2SO, 500 ng of HIV-1, or HIV-1 chimeras and 1 pmol of labeled HIV-1 U5 duplex substrate as indicated. Reaction mixtures were assembled from individual components and preincubated on ice overnight. To start the processing reaction, MgCl2 was added to a final concentration of 10 mm, and reaction mixtures were incubated at 37 °C for 90 min. The reactions were stopped by the addition of 2 μl of stop buffer (95% formamide, 20 mm EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue), heated at 95 °C for 5 min, and then placed on ice. Products of the reaction were separated through a 20% polyacrylamide denaturing gel. Labeled reaction products were visualized using Kodak MR film exposed overnight. For reactions containing ASV U5 or U3 LTR substrate, the final reaction mixture contained 20 mm MOPS, pH 7.2, 3 mm dithiothreitol, 100 μg/ml bovine serum albumin, 500 ng of ASV IN, and 1 pmol of labeled duplex substrates as indicated. Structural Model for HIV-1 IN with Bound LTR DNA—The initial model for the full-length three-domain IN structure was constructed using the separate two-domain crystal structures of the N-terminal domain and catalytic core (PDB code 1K6Y (49.Wang J.-Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar)) and the catalytic core and C-terminal domain (PDB code 1EX4 (50.Chen J.C. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci., U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar)). The two-domain structures were combined to form a three-domain structure by superimposing the catalytic core domains. The 1K6Y tetramer built from crystal lattice contacts was the basis for forming the tetramer of full-length IN. The zinc coordination complex was covalently modeled using 2.25-Å bond length restraints to the coordinating histidines and 2.35 Å to the coordinating cysteines. Residues 271–288 are disordered at the C terminus (50.Chen J.C. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci., U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar) and were omitted from the model. Residues 47–55 between the N-terminal and catalytic domains and the loop of residues 140–148 are disordered in the N-terminal two-domain structure (49.Wang J.-Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar). The missing loops were produced by obtaining initial Cα–Cα distances from a dynamic programming search of overlapping 30-mers generated from the whole PDB protein structure data base (51.Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27937) Google Scholar). The program AMMP (52.Harrison R.W. J. Comp. Chem. 1993; 14: 1112-1122Crossref Scopus (81) Google Scholar) was used with the current all-atom sp4 potential set (53.Weber I.T. Harrison R.W. Protein Eng. 1996; 9: 679-690Crossref PubMed Scopus (33) Google Scholar, 54.Weber I.T. Harrison R.W. Protein Sci. 1997; 11: 2365-2374Google Scholar). The charge generation parameters were taken from Bagossi et al. (55.Bagossi P. Zahuczky G. Tozser J. Weber I.T. Harrison R.W. J. Mol. Model. 1999; 5: 143-152Crossref Google Scholar). The new atoms were built using the Kohonen and analytic model-building features of AMMP (56.Harrison R.W. J. Math. Chem. 1999; 26: 125-137Crossref Google Scholar), minimized with conjugate gradients. The amortized fast multipole algorithm in AMMP was used for the long-range terms in the nonbonded and electrostatic potentials so that no cut-off radius was employed. The model contained 4 potassium and 4 phosphate ions from the crystal structures. The tetramer model was solvated in 16,195 water molecules. This solvated integrase was subjected to molecular dynamics simulation for 1.0 ns on a 1-GHz Linux PC. Frames were saved every 1 ps. The initial model for viral DNA was taken from the crystal structure of 1K61, which contained 20 base pairs of B-DNA. The DNA was converted to the HIV U5 sequence using the AMMP molecular mechanics and dynamics program (52.Harrison R.W. J. Comp. Chem. 1993; 14: 1112-1122Crossref Scopus (81) Google Scholar). AMMP was used to generate new atomic positions by means of an analytic coordinate generator (56.Harrison R.W. J. Math. Chem. 1999; 26: 125-137Crossref Google Scholar) followed by conjugate gradients minimization for each substituted base pair. Then the entire DNA molecule was minimized using conjugate gradients with all nonbonded and geometric terms. The DNA was positioned approximately using the DNA at the catalytic site of Tn5 transposase as a guide to place the processed 3′-OH at the catalytic site of IN. The two processed deoxyribonucleotides GT were removed from the 3′ end. A magnesium ion was placed between the 3′-OH, and the side chains of Asp64 and Asp116 at the catalytic site of each subunit. The DNA was rotated and translated into the groove formed between the catalytic domain of subunit B and the N- and C-terminal domains of subunit D approximately using the program O (57.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The fit to IN was optimized using the GDock routine of AMMP with harmonic restraints to atomic positions for critical atoms (tethers) and restraints on the DNA interatomic distances followed by conjugate gradients minimization. The GDock routine uses a genetic algorithm to search rotational and translational space where the rotational components are represented with unit quaternions. The use of unit quaternions instead of orthogonal rotation matrices avoids artificial singularities in the search space and greatly enhances the convergence of the algorithm. The two unpaired nucleotides 1 and 2 were moved out of the B-form using torsion searches to eliminate collisions with the protein. The 3′-OH was restrained to coordinate with the magnesium ion at the catalytic site. Once the first DNA was placed in the groove, the second LTR end was placed by symmetry in the adjacent groove between the catalytic domains of subunit D and the N- and C-terminal domains of subunit B followed by alternating rigid body movements of the LTR and C-terminal domain using GDock to overcome collisions (as the tetramer is not completely symmetric). Finally, the complex was minimized by conjugate gradients to ensure good nonbonded interactions. Structural Model of HIV-1 IN with LTR DNA—Although a crystal structure for full-length HIV-1 integrase is not yet available, we developed a model for an IN homotetramer bound to its LTR DNA substrate using knowledge of existing integrase two-domain structures, the retrotransposon transposase (Tc3 and Tn5), and the deduced amino acid sequence alignment of known retroviral integrases. Fig. 1 shows our structural model for HIV-1 integrase. We assembled the three functional domains of integrase into an integrated structure by superimposing the separate two-domain crystal structures for integrase: the N-terminal domain and catalytic core (PDB code 1K6Y (44.van Pouderoyen G. Ketting R.F. Perrakis A. Plasterk R.H. Sixma T.K. EMBO. 1997; 16: 6044-6054Crossref PubMed Scopus (91) Google Scholar, 49.Wang J.-Y. Ling H. Yang W. Craigie R. EMBO J. 2001; 20: 7333-7343Crossref PubMed Scopus (313) Google Scholar, 59.Yang Z. Mauser T. Bushman F. Hyde C. J. Mol. Biol. 2000; 296: 535-548Crossref PubMed Scopus (112) Google Scholar)) and the catalytic core and C-terminal domain (1EX4 (50.Chen J.C. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci., U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar)) (Fig. 1A). A tetramer model of IN was assembled using crystal lattice contacts (Fig. 1B). In this model the N- and C-terminal domains were arranged on the same side of the subunit and lay relatively close to each other. Molecular dynamics simulations of the solvated IN tetramer model were used to explore the conformational variation of the three domains in the
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