Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor

Łubkowski, J.; Yang, Fan; Alexandratos, Jerry; Wlodawer, Alexander; Zhao, He; Burke, Terrence R.; Neamati, Nouri; Pommier, ﻿Yves; Merkel, George; Skalka, Anna Marie

doi:10.1073/pnas.95.9.4831

Cited by 81 publications

(95 citation statements)

References 35 publications

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“…These residues were therefore proposed to directly bind the required metal cofactors. Later studies showed that divalent cations, such as Ca Binding studies of human immunodeficiency virus type 1 IN and ASV IN with an inhibitor targeted against human immunodeficiency virus type 1 IN showed comparable inhibition effects, but the crystal structure of an inhibitor-catalytic core domain complex was determined only for ASV IN (8). One interesting observation during that study was the rotation of the side chain of the central acidic residue, Asp-64, around the C␣OC␤ bond.…”

Section: Integrase (In)mentioning

confidence: 97%

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: Integrase (In)mentioning

confidence: 97%

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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“…In our previous reports (21,22), we noted a new conformation of the active-site loop, as well as vastly different conformations of the side chain of Asp64; therefore, in the present study, we sought to minimize model bias. The refinement was cross-validated by the R free index (24), which was calculated using 10% of all reflections.…”

Section: Methodsmentioning

confidence: 99%

“…Only two conserved water molecules interact with the protein via a single hydrogen bond. About one-fourth of these conserved water molecules (21) are located within the crystallographic dimer interface, and four of them (Wat402, Wat413, Wat470, and Wat472) clearly play a direct role in stabilizing dimerization.…”

Section: Structures Of Asvin-trn and D64n-trnmentioning

confidence: 99%

“…Crystal structures of the catalytic core domain of ASV IN have also been published with divalent metal cations bound in the active site (15,20), as well as with an inhibitor of HIV-1 IN (21). The active-site loop of ASV IN was shown to accommodate different conformations depending on the crystallization conditions (14,20), although the most recent reports discussed difficulties in finding stable conformations of this loop (21,22).…”

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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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“…Knowledge of key amino acid residues involved in the binding of potential drugs, and therefore which residues are likely to mutate under therapeutic pressure, would inevitably help researchers stay one step ahead of drug-resistant viral strains. A co-crystal structure of one of our inhibitors was previously solved with the ASV-IN (4,5). This complex was subsequently used as a surrogate structure to discover IN inhibitors through highthroughput docking studies (6).…”

mentioning

confidence: 99%

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

Al‐Mawsawi

Fikkert

Dayam

et al. 2006

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

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Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photocrosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128 AACWWAGIK 136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling͞MS analysis with site-directed mutagenesis͞molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.drug design ͉ mass spectrometry ͉ photoaffinity labeling H IV-1 integrase (IN) mediates the insertion of viral DNA into the host genome. This process occurs through two separate events, both catalyzed by IN. In the 3Ј-processing reaction, IN cleaves a dinucleotide adjacent to a conserved CA on each terminus of the reverse-transcribed viral DNA. This cleavage results in two 3Ј hydroxyl groups that are used for a subsequent nucleophilic attack. IN then inserts this DNA product into the host genome in the second reaction, termed strand transfer (1, 2). IN reactions can be carried out in vitro by using purified protein, a DNA substrate with ends mimicking the U3 or U5 viral DNA termini, and Mg 2ϩ or Mn 2ϩ as a cofactor (3).Structural information detailing the association between IN and inhibitors under development is of enormous therapeutic importance. Knowledge of key amino acid residues involved in the binding of potential drugs, and therefore which residues are likely to mutate under therapeutic pressure, would inevitably help researchers stay one step ahead of drug-resistant viral strains. A co-crystal structure of one of our inhibitors was previously solved with the ASV -IN (4, 5). This complex was subsequently used as a surrogate structure to discover IN inhibitors through highthroughput docking studies (6). Thus far, only two examples of co-crystal structures of HIV-1 IN core in complex with inhibitors have been reported (7,8). Despite our own repeated attempts, solving co-crystal structures of IN with our potent inhibitors has failed. This pauc...

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Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor

Cited by 81 publications

References 35 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

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

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

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Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor

Cited by 81 publications

References 35 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

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

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

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