Mapping the Epitope of an Inhibitory Monoclonal Antibody to the C-terminal DNA-binding Domain of HIV-1 Integrase

Yi, Jizu; Cheng, Hong; Andrake, Mark D.; Dunbrack, Roland L.; Röder, Heinrich; Skalka, Anna Marie

doi:10.1074/jbc.m105072200

Cited by 21 publications

(23 citation statements)

References 43 publications

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“…Our HIV-1 IN model predicts that binding of either one of these antibodies would interfere with the assembly of a functional reaching dimer. Furthermore, alanine substitution of any one of these epitope residues drastically reduces the single end joining activity of HIV-1 IN (53,60). This result is consistent with the derived reaching dimer interface of HIV-1 IN in which Lys-34 is predicted to hydrogen bond with Glu-246; buried Arg-262 is predicted to interact with the backbones of Pro-30 and Val-31 and Arg-263 to hydrogen bond with Glu-33.…”

Section: Discussionsupporting

confidence: 75%

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Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Bojja

Andrake

Weigand

et al. 2011

Journal of Biological Chemistry

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We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly. Retroviral integrase (IN)3 catalyzes the insertion of viral DNA into the DNA of the infected host cell. IN is one of three retrovirally encoded enzymes that are essential for retroviral replication and therefore is an important target for drugs to treat HIV/AIDS. One IN active site-directed inhibitor, raltegravir, is approved by the Food and Drug Administration for this purpose, and a second, elvitegravir, is in advanced clinical trials.

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Section: Discussionsupporting

confidence: 75%

“…Previous studies on the mechanism of inhibition by monoclonal antibodies that are specific to the HIV-IN NTD and CTD have uncovered critical epitope residues in these domains (59,60). The inhibitory activity of mAb17 can be explained by distortion of the NTD helix-turn-helix motif via binding to residues 25-35.…”

Section: Discussionmentioning

confidence: 99%

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Bojja

Andrake

Weigand

et al. 2011

Journal of Biological Chemistry

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“…It is suggested that such a binding affinity difference might be due to the ability of CCD to bind viral DNA and the nonspecific DNA binding characteristic of CTD for the full length HIV-1 IN. [23,24] Therefore, our SPR-based result that HIV-1 NTD could bind to viral DNA supports the previously published suggestion that NTD can bind viral DNA [25] and implies that the process of preintegrated complex formation of IN possibly involves the direct interaction between NTD and viral DNA, in addition to viral DNA-CCD binding. This result thereby confirms that hyrtiosal might inhibit HIV-1 IN interaction with viral DNA through its binding to NTD.…”

Section: Resultssupporting

confidence: 88%

Hyrtiosal, from the Marine Sponge Hyrtios erectus, Inhibits HIV‐1 Integrase Binding to Viral DNA by a New Inhibitor Binding Site

Shen

et al. 2008

ChemMedChem

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HIV-1 integrase (IN) is composed of three domains, the N-terminal domain (NTD, residues 1-50), the catalytic core domain (CCD, residues 51-212), and the C-terminal domain (CTD, residues 213-288). All the three domains are required for the two known integration reactions. CCD contains the catalytic triad and is believed to bind viral DNA specifically, and CTD binds viral DNA in a nonspecific manner. As no clear evidence has confirmed the involvement of NTD in DNA binding directly, NTD has not been seriously considered and less is known about its function in viral replication. In the current work, using a SPR technology-based assay, the HIV-1 viral DNA was determined to bind directly to NTD with a K(D) value of 8.8 microM, suggesting that the process of preintegrated complex formation for HIV-1 IN might involve the direct interaction of NTD with viral DNA in addition to binding of viral DNA to the catalytic core domain and C-terminal domain. Moreover, such viral DNA/IN binding could be inhibited by the marine product hyrtiosal from the marine sponge Hyrtios erectus with an IC(50) of 9.60+/-0.86 microM. Molecular dynamic analysis correlated with a site-directed mutagenesis approach further revealed that such hyrtiosal-induced viral DNA/IN binding inhibition was caused by the fact that hyrtiosal could bind HIV-1 NTD at Ser17, Trp19, and Lys34. As hyrtiosal was recently discovered by us as a protein tyrosine phosphatase 1B (PTP1B) inhibitor,1 this work might also supply multiple-target information for this marine product, and the verified HIV-NTD/HIV-1 IN interaction model could have further implications for new HIV-1 IN inhibitor design and evaluation.

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“…A large molecule inhibitor, monoclonal antibody 33, was reported to bind to the C-terminal domain and impair IN coordination of the cognate DNA (17). Crystallographic and NMR efforts to determine small molecule inhibitor binding sites were restricted to analyses of the protein core domain rather than full- length IN (23, 24).…”

Section: Resultsmentioning

confidence: 99%

Mass Spectrometric Analysis of the HIV-1 Integrase-Pyridoxal 5′-Phosphate Complex Reveals a New Binding Site for a Nucleotide Inhibitor

Williams

Zhang

Shkriabai

et al. 2005

Journal of Biological Chemistry

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HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5-phosphate (PLP).The continuing emergence of new HIV-1 1 variants resistant to current therapeutic treatments, which include small molecule inhibitors targeting HIV-1 reverse transcriptase and protease, makes the search for new anti-HIV-1 drugs imperative. In this regard, HIV-1 integrase (IN), which has no known human counterparts, is an attractive target. Furthermore, the fact that IN uses a common active site for 3Ј-processing and DNA strand transfer may constrain the range of mutations that can contribute to evolution of viable drug-resistant viruses.HIV-1 IN catalyzes integration of the viral DNA, made by reverse transcription, into the host chromosome in a two-step reaction (reviewed in Ref. 1). In the first step, called 3Ј-processing, two nucleotides are removed at each 3Ј-end of the viral DNA. In the next step, called DNA strand transfer, concerted transesterification reactions integrate the viral DNA ends into the host genome.HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (residues 1-50) that contains the HHCC zinc-binding motif, the core domain (residues 51-212) that contains the catalytic site, and the C-terminal domain (residues 213-270) that is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (2-6). In addition, the structures of the combined core and C-terminal domains (7) and core and N-terminal domains (8) have been recently determined. However, efforts to obtain a structure of the full-length protein have been impeded by poor protein solubility.Purified IN-based assays have been employed for screening of potential inhibitors. These studies have revealed several classes of compounds with anti-HIV-1 IN activity, including diketo acids, naphthyridines, pyranodipyrimidines, nucleotide analogs, hydroxylated aromatic compounds, DNA-interacting agents, peptides, and antibodies (9 -22). Much effort has been devoted to dissecting the mechanism of inhibition and identifying the inhibitor binding sites. Crystallographic studies revealed two distinct binding sites in IN including the catalytic site for a diketo group-containing inhibitor (1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone) and a site located near the IN dimer interface for 3,4-dihydroxyphenyltriphenylarsonium bromide (23,24). However, these studies employed the isolated core domain of IN rather then the fulllength protein.Mass spectrometry is a powerful structural biology tool that allows the analysis of protein-inhibitor complexes under biologically relevant conditions and provides structural information complementary to NMR and crystallography. For example, we recently reported identification of a small molecule inhibitor binding site to full-length IN using...

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Mapping the Epitope of an Inhibitory Monoclonal Antibody to the C-terminal DNA-binding Domain of HIV-1 Integrase

Cited by 21 publications

References 43 publications

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

Hyrtiosal, from the Marine Sponge Hyrtios erectus, Inhibits HIV‐1 Integrase Binding to Viral DNA by a New Inhibitor Binding Site

Mass Spectrometric Analysis of the HIV-1 Integrase-Pyridoxal 5′-Phosphate Complex Reveals a New Binding Site for a Nucleotide Inhibitor

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