The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations

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

doi:10.1016/s0969-2126(96)00012-3

Cited by 164 publications

(146 citation statements)

References 32 publications

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“…Both observed conformations of this side chain appear to be energetically favorable. In the first mode of binding, predominantly observed in the previously reported structures (6,7,9), Asp-64 is anchored by a hydrogen bond to a water molecule, which in turn is stabilized by an additional hydrogen bond with the side chain of Asp-121. Because it was shown previously that this conformation of Asp-64 remains unchanged upon binding of several divalent metal cations, we refer to it as the "active" conformation.…”

Section: Discussionmentioning

confidence: 92%

“…In the previously reported structures of ASV IN (6,7,9), with the sole exception of the low pH complexes with an inhibitor (8), the side chain of Asp-64 points toward the side chain of Asp-121, forming hydrogen bonds mediated by a water molecule (or a metal ion). The difference F o Ϫ F c electron density extending from C␤ of residue 64 unambiguously indicates that the Asx-64 side chain (Asn in the mutant ASVIN64 or Asp in ASVIN5) is rotated by ϳ150°around the C␣OC␤ bond (Fig.…”

Section: Conformation Of Asp/asn-64 -mentioning

confidence: 99%

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Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Łubkowski¹,

Yang²,

Alexandratos³

et al. 1998

Journal of Biological Chemistry

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Crystallographic studies of the catalytic core domain of avian sarcoma virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs against AIDS. Recently, crystals of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations. Data were also collected at low pH for the crystals of the native ASV IN core domain. In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx-64 (Asx denotes Asn or Asp) is rotated by ϳ150°around the C␣OC␤ bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn 2؉ can still bind to Asp-121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner. Integrase (IN)1 (1) is one of only four enzymes encoded by retroviruses, such as human immunodeficiency virus type 1 and avian sarcoma virus (ASV), and it is absolutely essential for the support of the viral life cycle. For these reasons, IN is currently a target for the design of antiretroviral drugs. Retroviral INs contain approximately 300 amino acids, organized into three domains: zinc-binding N terminus, catalytic core domain, and DNA-binding C terminus. IN catalyzes the incorporation of the reverse-transcribed viral DNA into the host genome in two steps, processing and joining (1-3), both of which involve a 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, two nucleotides from the 3Ј-ends of both viral DNA strands. In the joining step, the exposed viral DNA deoxyribose 3Ј-OH is activated to attack the host DNA at a relatively nonspecific location, thereby inserting viral DNA into the host genome. In vitro, these reactions require only virus-like DNA, IN, and metal cations.Although the isolated ASV IN catalytic core domain is defective for the processing and joining activities, it retains two activities: disintegration, the reverse of the joining step that uses a preformed DNA substrate, and an endonuclease activity that cleaves between the highly conserved C and A (Ϫ3 activity) at the termini of the viral DNA (CATT-3Ј) (4). The endonuclease activity is distinct from the normal processing activity of IN, which cleaves between the A and T (Ϫ2 activity). Both the endonuclease and disintegration activities of the catalytic domain are dependent on the D, D(35)E catalytic triad, corresponding to Asp-...

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Section: Discussionmentioning

confidence: 92%

Section: Conformation Of Asp/asn-64 -mentioning

confidence: 99%

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Łubkowski¹,

Yang²,

Alexandratos³

et al. 1998

Journal of Biological Chemistry

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“…All IN DDE mutants display reduced 3Ј end processing, strand transfer, and disintegration, and mutation of any of these residues in related transposases and polynucleotidyltransferases diminishes their respective activities. Structures have been solved for HIV-1 and avian sarcoma virus (ASV) IN in the absence of DNA (14)(15)(16)(17)30) and for Tn5 transposase with a bound donor end (30). Crystals of the HIV-1 and ASV IN core domains with Mg 2ϩ or Mn 2ϩ contain one metal coordinated by D64 and D116 (site I; positions in HIV-1 IN); however, an additional metal coordinated by D64 and E152 (site II) was observed in ASV IN by using either Zn 2ϩ or Cd 2ϩ (17).…”

Section: Discussionmentioning

confidence: 99%

“…For IN and related enzymes that catalyze phosphoryl transfer reactions, the number of metals present and required in the active site remains controversial. Structural studies of IN reveal a single binding site for Mg 2ϩ or Mn 2ϩ (14)(15)(16), although a second metal has been observed with Zn 2ϩ or Cd 2ϩ (17).…”

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confidence: 99%

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Grobler

Stillmock²,

Hu³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

378

325

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The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3 end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metaldependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg 2؉ . The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.A n essential step in HIV replication is the integration of the reverse-transcribed viral genome into host chromosomal DNA by the virally encoded integrase (IN) protein (1-3). Integration is required for efficient long terminal repeat-driven transcription of the provirus for the production of viral proteins and RNA progeny. IN represents an important chemotherapeutic target, as its inactivation, either by mutagenesis or inhibition, blocks productive infection by HIV-1 (4-7).Integration is carried out in the cell in a series of distinct steps (8-10). First, IN cleaves the two terminal nucleotides from each 3Ј end of the viral DNA. The 3Ј processing reaction is carried out concurrently with or soon after reverse transcription in the cytoplasm. In the second step, strand transfer, IN catalyzes staggered nicking of the target chromosomal DNA and joining of each 3Ј end of the viral DNA to the 5Ј ends of the host DNA. Strand transfer is temporally and spatially separated from 3Ј processing and occurs after transport of the preintegration complex from the cytoplasm into the nucleus.Divalent metals such as Mg 2ϩ or Mn 2ϩ are required for both 3Ј processing and strand transfer and for the assembly of IN onto specific viral donor DNA to form a complex competent to carry out either function (11)(12)(13) (18), L-731,988 and related DKAs inhibit integration and viral replication in cell culture (7). Mutations that confer resistance to DKAs have been identified and map to IN residues adjacent to D64 and E152. The in...

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“…This motif is believed to be conserved among all transposases and retroviral integrases. The DDE residues serve to coordinate divalent metal ions that are required for catalysis (11)(12)(13)(14). A previous study has suggested that Asp-97 and Glu-326 contribute to the coordination of one metal ion, whereas Asp-97 and Asp-188 coordinate a second ion (15).…”

Section: Tn5 Transposase (Tnp)mentioning

confidence: 99%

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

Peterson

Reznikoff

2003

Journal of Biological Chemistry

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Tn5 transposase (Tnp), a 53.3-kDa protein, enables the movement of transposon Tn5 by a conservative mechanism. Within the context of a protein and DNA synaptic complex, a single Tnp molecule catalyzes four sequential DNA breaking and joining reactions at the end of a single transposon. The three amino acids of the DDE motif (Asp-97, Asp-188, and Glu-326), which are conserved among transposases and retroviral integrases, have been shown previously to be absolutely required for all catalytic steps. To probe the effect of active site geometry on the ability to form synaptic complexes and perform catalysis, single mutations at each position of the DDE motif were constructed. The aspartates were changed to glutamates, and the glutamate was changed to an aspartate. These mutants were studied by performing in vitro binding assays using short oligonucleotide substrates simulating the natural substrates for the synaptic complex formation and subsequent transposition steps. The results indicate that the aspartate to glutamate mutations restrict synaptic complex formation with substrates resembling the natural transposon prior to transferred strand nicking. This suggests a structural model in which the donor backbone DNA, prior to nicking, occupies the same space that is invaded by the longer side chains present in the aspartate to glutamate mutants. Additionally, catalytic assays support the previous proposal that the active site coordinates two divalent metal ions.

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The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations

Cited by 164 publications

References 32 publications

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

Tn5 Transposase Active Site Mutations Suggest Position of Donor Backbone DNA in Synaptic Complex

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