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      A Novel Co-Crystal Structure Affords the Design of Gain-of-Function Lentiviral Integrase Mutants in the Presence of Modified PSIP1/LEDGF/p75

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          Abstract

          Lens epithelium derived growth factor (LEDGF), also known as PC4 and SFRS1 interacting protein 1 (PSIP1) and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN) proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD) of HIV-2 IN in complex with the IN binding domain (IBD) of LEDGF. The structure extends the known IN–LEDGF interface, elucidating primarily charge–charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD–IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN–LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy.

          Author Summary

          Retroviruses crucially rely on insertion of their genomes into a host cell chromosome, and this process is carried out by the viral enzyme integrase. HIV and other lentiviruses also depend on LEDGF, a cellular chromatin-associated protein, which binds their integrase proteins and tethers them to a human chromosome. The interaction between integrase and LEDGF can potentially be exploited for directing integration of lentiviral vectors in gene therapy applications, as well as for development of antiretroviral drugs. Herein, we present a three-dimensional structure of a protein–protein complex containing a fragment of HIV integrase and the integrase-binding domain of LEDGF. Our structure elucidates the hitherto unknown LEDGF–integrase interface involving the amino terminal portion of the viral enzyme. Using a range of complementary approaches, we further show that these novel protein–protein contacts are essential for the function of LEDGF in HIV integration. The novel structural details will be very useful for the development of HIV inhibitors that target the integrase–LEDGF interaction. Furthermore, they enabled us to design a mutant of HIV integrase that depends on a reverse-engineered mutant of LEDGF, providing an inroad to the design of LEDGF-based lentiviral vector targeting strategies.

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          Most cited references33

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          Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.

          DNA and a large proportion of RNA are antiparallel duplexes composed of an unvarying phosphosugar backbone surrounding uniformly stacked and highly similar base pairs. How do the myriad of enzymes (including ribozymes) that perform catalysis on nucleic acids achieve exquisite structure or sequence specificity? In all DNA and RNA polymerases and many nucleases and transposases, two Mg2+ ions are jointly coordinated by the nucleic acid substrate and catalytic residues of the enzyme. Based on the exquisite sensitivity of Mg2+ ions to the ligand geometry and electrostatic environment, we propose that two-metal-ion catalysis greatly enhances substrate recognition and catalytic specificity.
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            Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast.

            Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast. The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins. Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation. Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast.
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              Menin critically links MLL proteins with LEDGF on cancer-associated target genes.

              Menin displays the unique ability to either promote oncogenic function in the hematopoietic lineage or suppress tumorigenesis in the endocrine lineage; however, its molecular mechanism of action has not been defined. We demonstrate here that these discordant functions are unified by menin's ability to serve as a molecular adaptor that physically links the MLL (mixed-lineage leukemia) histone methyltransferase with LEDGF (lens epithelium-derived growth factor), a chromatin-associated protein previously implicated in leukemia, autoimmunity, and HIV-1 pathogenesis. LEDGF is required for both MLL-dependent transcription and leukemic transformation. Conversely, a subset of menin mutations in multiple endocrine neoplasia type 1 patients abrogate interaction with LEDGF while preserving MLL interaction but nevertheless compromise MLL/menin-dependent functions. Thus, LEDGF critically associates with MLL and menin at the nexus of transcriptional pathways that are recurrently targeted in diverse diseases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                January 2009
                January 2009
                9 January 2009
                : 5
                : 1
                : e1000259
                Affiliations
                [1 ]Division of Medicine, Imperial College London, St. Mary's Campus, London, United Kingdom
                [2 ]Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America
                University of Pennsylvania School of Medicine, United States of America
                Author notes
                [¤]

                Current address: School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia

                Conceived and designed the experiments: SH AE PC. Performed the experiments: SH MCS SSG EV AE PC. Analyzed the data: SH MCS SSG AE PC. Wrote the paper: SH AE PC.

                Article
                08-PLPA-RA-1212R2
                10.1371/journal.ppat.1000259
                2606027
                19132083
                541805e0-02a2-4240-bb48-cd95e834df69
                Hare et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 9 October 2008
                : 8 December 2008
                Page count
                Pages: 12
                Categories
                Research Article
                Biochemistry
                Molecular Biology
                Virology/Immunodeficiency Viruses

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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