High-resolution Structure of the Catalytic Domain of Avian Sarcoma Virus Integrase

Bujacz, G.; Jaskólski, M.; Alexandratos, Jerry; Wlodawer, Alexander; Merkel, George; Katz, Richard A.; Skalka, A M

doi:10.1006/jmbi.1995.0556

Cited by 219 publications

(222 citation statements)

References 45 publications

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“…The atomic resolution models of ASV IN presented here provide a number of new structural details and an overall unparalleled accuracy in terms of both atomic position and thermal motion description, compared with the previous reports (14,15,20,22). Because the structure of ASV IN under different crystallization conditions has been described previously, only the new observations will be discussed here in detail.…”

Section: Resultsmentioning

confidence: 87%

“…Most of these structures, with the exception of ASVIN-TRN and D64N-TRN, were described previously at lower resolution (14,22). Therefore, only rigid body refinement was necessary for obtaining starting models.…”

Section: Methodsmentioning

confidence: 99%

“…Crystals of the core domain of ASV IN were grown according to protocols described previously (14), using either PEG4000 or ammonium sulfate as the precipitant. In the case of the D64N derivative, only crystals grown in the presence of PEG4000 were studied.…”

Section: Methodsmentioning

confidence: 99%

“…As described previously (14), ASV IN crystals grown in the presence of Hepes (the condition for both of the highpH structures described here) bind this molecule in a specific fashion. Such binding triggers minor structural changes that are not directly pH dependent.…”

Section: Comparison Of the Structures Of Asv Inmentioning

confidence: 99%

“…The originally published structure of the catalytic core domain of HIV-1 IN lacked a number of residues in the active-site area (one catalytic residue and the entire flexible active-site loop) due to their disorder, and the conformations of the other side chains in the active site (most notably Asp64 and Glu116) diverged significantly from those found in the related enzymes (14)(15)(16). These distor-tions of the active site were the most likely cause of the inability of crystallized HIV-1 IN to bind divalent cations.…”

mentioning

confidence: 99%

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Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

et al. 1999

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Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 f Asn substitution have been solved at atomic resolution ranging 1.02-1.42 Å. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.Retroviruses, such as human immunodeficiency virus type 1 (HIV-1) 1 or avian sarcoma virus (ASV), encode in their genes three essential enzymes: reverse transcriptase (RT), protease (PR), and integrase (IN) (1). In rare cases a retrovirus such as feline immunodeficiency virus and equine infectious anemia virus also encodes dUTP-ase (2-4). The first three enzymes are considered to be primary targets for designing drugs against AIDS, because each enzyme is absolutely required for virus replication. Although the search for therapeutically suitable inhibitors has been successful for RT and PR and a number of drugs against these enzymes are already in use (5), no such drugs targeted against IN are yet available. This is due in part to the lack of a complete structural description of IN, which is crucial for understanding its enzymatic activity. Such knowledge is the foundation of rational drug design (6).A molecule of retroviral IN contains approximately 300 amino acids, and it comprises three domains: the zincbinding N-terminal domain, the catalytic core domain, and the DNA-binding C-terminal domain. IN catalyzes the incorporation of reverse-transcribed viral DNA into the host genome in two steps, processing and joining (1,7,8), both of which involve nucleophilic attack by a hydroxyl group on a DNA phosphate. In the processing step, a water molecule attacks near the end of the viral DNA, displacing two nucleotides from the 3′ ends of each of the viral DNA strands. In the joining step, each exposed viral DNA ribose 3′-OH is activated to attack the host DNA at a relatively nonspecific location with a separation of five or six nucleotides, thereby inserting viral DNA into the host genome. In vitro, these reactions require only IN, metal cations, and DNA, although other proteins play a role in vivo.The structures of the N-terminal domain (9, 10) and C-terminal domain (11, 12) of HIV IN have been determined by nuclear magnetic resonance (NMR) spectroscopy, whereas the structure of the catalytic core domain ha...

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Section: Resultsmentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Comparison Of the Structures Of Asv Inmentioning

confidence: 99%

mentioning

confidence: 99%

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Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

et al. 1999

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Refined solution structure of the c-terminal DNA-binding domain of human immunovirus-1 integrase

et al. 1999

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The structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well-defined dimer interface. The folding topology of the monomer consists of a five-stranded beta-barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back-bone root mean square deviation versus the average of 0.46 A. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556-564.

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Similarities in the HIV-1 and ASV integrase active sites upon metal cofactor binding

2000

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The HIV‐1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. We report here a fully hydrated 2 ns molecular dynamics simulation performed using parallel NWChem3.2.1 with the AMBER95 force field. The HIV‐1 integrase catalytic domain previously determined by crystallography (1B9D) and modeling including two Mg2+ ions placed into the active site based on an alignment against an ASV integrase structure containing two divalent metals (1VSH), was used as the starting structure. The simulation reveals a high degree of flexibility in the region of residues 140–149 even in the presence of a second divalent metal ion and a dramatic conformational change of the side chain of E152 when the second metal ion is present. This study shows similarities in the behavior of the catalytic residues in the HIV‐1 and ASV integrases upon metal binding. The present simulation also provides support to the hypothesis that the second metal ion is likely to be carried into the HIV‐1 integrase active site by the substrate, a strand of DNA. © 2000 John Wiley & Sons, Inc. Biopoly 53: 308–315, 2000

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High-resolution Structure of the Catalytic Domain of Avian Sarcoma Virus Integrase

Cited by 219 publications

References 45 publications

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

Refined solution structure of the c-terminal DNA-binding domain of human immunovirus-1 integrase

Similarities in the HIV-1 and ASV integrase active sites upon metal cofactor binding

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High-resolution Structure of the Catalytic Domain of Avian Sarcoma Virus Integrase

Cited by 219 publications

References 45 publications

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant,

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant,

Refined solution structure of the c-terminal DNA-binding domain of human immunovirus-1 integrase

Similarities in the HIV-1 and ASV integrase active sites upon metal cofactor binding

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Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,