Integration Requires a Specific Interaction of the Donor DNA Terminal 5′-Cytosine with Glutamine 148 of the HIV-1 Integrase Flexible Loop

Johnson, Allison A.; Santos, Webster L.; Pais, Godwin C. G.; Marchand, Christophe; Amin, Ronak; Burke, Terrence R.; Verdine, Gregory; Pommier, ﻿Yves

doi:10.1074/jbc.m511348200

Cited by 67 publications

(63 citation statements)

References 45 publications

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“…Preparation of Recombinant HIV-1 IN, DNA Oligonucleotides, and Cross-linked Products-Full-length IN and mutant proteins were expressed in E. coli and purified as described previously (35). The catalytic activities of recombinant proteins were monitored according to the reported procedure (35).…”

Section: Methodsmentioning

confidence: 99%

“…The catalytic activities of recombinant proteins were monitored according to the reported procedure (35).…”

Section: Methodsmentioning

confidence: 99%

“…Post-synthetic attachment of the carbon tether (cystamine) to the modified bases, and subsequent activation and purification of oligonucleotides were performed as published previously (35).…”

Section: Methodsmentioning

confidence: 99%

“…Site directed mutagenesis experiments indicated that different monomers within the IN multimer provide complementary rather than symmetrical contacts to DNA (29 -31). Photo and chemical cross-linking studies revealed several potential DNA binding residues (32)(33)(34)(35)(36)(37). These biochemical experiments together with crystallographic determination of the two-domain structures prompted molecular modeling research.…”

Section: Hiv-1 Integrase (In)mentioning

confidence: 99%

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Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding

Zhao

McKee

Kessl

et al. 2008

Journal of Biological Chemistry

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To identify functional contacts between HIV-1 integrase (IN)and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys-and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The footprinting studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes. HIV-1 integrase (IN)4 is commonly viewed as an important therapeutic target for the following reasons: its catalytic activities are required for viral replication, there is no closely related cellular equivalent of IN, and specific IN inhibitors are likely to be effective against viral strains resistant to currently available therapies targeting reverse transcriptase (RT), protease, and virus-cell fusion. Detailed structural information on functional IN-DNA complexes could aid drug design efforts. For example, the promising diketo acid class of inhibitors preferentially bind to the assembled IN-viral DNA complex rather than the free protein (1-4).The two chemical reactions catalyzed by HIV-1 IN, 3Ј processing and DNA strand transfer, have been characterized in detail (reviewed in Ref. 5). First, IN removes two nucleotides from each 3Ј-end of the viral DNA synthesized by reverse transcriptase. In the following step, concerted transesterification reactions covalently join the viral DNA ends into the host genome (6). In vivo, the enzyme acts in the context of a large nucleoprotein complex with a number of viral and host proteins contributing to the integration process (7-21).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (NTD) (residues 1-50) that contains an HHCC zinc binding motif, the catalytic core domain (CCD) (residues 51-212) containing the DDE motif essential for coordinating catalytic divalent metals, and the C-terminal domain (CTD) (residues 213-288) that is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (22-26). In addition, two-domain CCD/ CTD (27) and NTD/CCD (28) crystal structures have been determined. However,...

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Section: Methodsmentioning

confidence: 99%

“…The catalytic activities of recombinant proteins were monitored according to the reported procedure (35).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Hiv-1 Integrase (In)mentioning

confidence: 99%

See 2 more Smart Citations

Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding

Zhao

McKee

Kessl

et al. 2008

Journal of Biological Chemistry

Self Cite

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“…The molecule dynamics simulation results of full-length integrase-DNA complex suggested that the catalytic loop played crucial role during integration process (De Luca et al, 2005). It had been reported that the residue Gln148 of HIV-1 integrase was critical for strand transfer (Johnson et al, 2006).…”

Section: Comparison Of the Two Different Dimersmentioning

confidence: 99%