Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor
2009; Springer Nature; Volume: 28; Issue: 7 Linguagem: Inglês
10.1038/emboj.2009.41
ISSN1460-2075
AutoresFabrice Michel, Corinne Crucifix, Florence Granger, Sylvia Eiler, Jean‐François Mouscadet, S.V. Korolev, Julia Agapkina, Rustam Ziganshin, Marina Gottikh, Alexis Nazabal, Stéphane Emiliani, Richard Benarous, Dino Moras, Patrick Schultz, Marc Ruff,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle19 February 2009free access Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor Fabrice Michel Fabrice Michel IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Corinne Crucifix Corinne Crucifix IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Florence Granger Florence Granger IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Sylvia Eiler Sylvia Eiler IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Jean-François Mouscadet Jean-François Mouscadet Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, CNRS, ENS-Cachan, Institut d'Alembert, Cachan, France Search for more papers by this author Sergei Korolev Sergei Korolev Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Julia Agapkina Julia Agapkina Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Rustam Ziganshin Rustam Ziganshin Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Marina Gottikh Marina Gottikh Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Alexis Nazabal Alexis Nazabal Department of Chemistry and Applied Biosciences ETH Zurich and CovalX, Technoparkstrasse, Zürich, Switzerland Search for more papers by this author Stéphane Emiliani Stéphane Emiliani Institut Cochin, Département des Maladies Infectieuses, Université Paris Descartes, CNRS, Paris, France Inserm, Paris, France Search for more papers by this author Richard Benarous Richard Benarous CellVir, Evry, France Search for more papers by this author Dino Moras Dino Moras IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Patrick Schultz Corresponding Author Patrick Schultz IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Marc Ruff Corresponding Author Marc Ruff IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Fabrice Michel Fabrice Michel IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Corinne Crucifix Corinne Crucifix IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Florence Granger Florence Granger IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Sylvia Eiler Sylvia Eiler IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Jean-François Mouscadet Jean-François Mouscadet Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, CNRS, ENS-Cachan, Institut d'Alembert, Cachan, France Search for more papers by this author Sergei Korolev Sergei Korolev Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Julia Agapkina Julia Agapkina Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Rustam Ziganshin Rustam Ziganshin Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Marina Gottikh Marina Gottikh Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Alexis Nazabal Alexis Nazabal Department of Chemistry and Applied Biosciences ETH Zurich and CovalX, Technoparkstrasse, Zürich, Switzerland Search for more papers by this author Stéphane Emiliani Stéphane Emiliani Institut Cochin, Département des Maladies Infectieuses, Université Paris Descartes, CNRS, Paris, France Inserm, Paris, France Search for more papers by this author Richard Benarous Richard Benarous CellVir, Evry, France Search for more papers by this author Dino Moras Dino Moras IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Patrick Schultz Corresponding Author Patrick Schultz IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Marc Ruff Corresponding Author Marc Ruff IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France Search for more papers by this author Author Information Fabrice Michel1, Corinne Crucifix1, Florence Granger1, Sylvia Eiler1, Jean-François Mouscadet2, Sergei Korolev3, Julia Agapkina3, Rustam Ziganshin3, Marina Gottikh3, Alexis Nazabal4, Stéphane Emiliani5,6, Richard Benarous7, Dino Moras1, Patrick Schultz 1 and Marc Ruff 1 1IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France 2Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, CNRS, ENS-Cachan, Institut d'Alembert, Cachan, France 3Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia 4Department of Chemistry and Applied Biosciences ETH Zurich and CovalX, Technoparkstrasse, Zürich, Switzerland 5Institut Cochin, Département des Maladies Infectieuses, Université Paris Descartes, CNRS, Paris, France 6Inserm, Paris, France 7CellVir, Evry, France *Corresponding authors. Department of Structural Biology and Genomics, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, Illkirch 67404, France. Tel.: +33 0388 6557 50; Fax: +33 0388 6532 76; E-mail: [email protected] or Tel.: +33 0388 6533 50; Fax: +33 0388 6532 76; E-mail: [email protected] The EMBO Journal (2009)28:980-991https://doi.org/10.1038/emboj.2009.41 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies. Introduction The human immunodeficiency virus (HIV-1) integrase (IN) catalyses the stable integration of the viral cDNA into the host genome, an essential step in the establishment of HIV infection. IN is a DNA recombinase that catalyses two endonucleolytic reactions. In the 3′-processing reaction, IN cleaves a dinucleotide from the U5 and U3 ends of viral cDNA, thereby exposing a 3′-OH group at each end of the viral DNA. In the strand-transfer reaction, IN generates a double-strand break in the host DNA and joins the newly formed ends to the viral 3′ ends by transesterification (Engelman et al, 1991). Host DNA repair proteins remove the two nucleotide overhang and fill in the DNA gaps to complete the integration reaction (Yoder and Bushman, 2000). Both reactions can be carried out in vitro with purified IN and DNA substrates that mimic the viral DNA ends, in the presence of cofactors such as Mg2+ or Mn2+ (Zheng et al, 1996). The enzyme consists of three structural and functional domains. The N-terminal zinc-binding domain (residues 1–50), which is required for 3′ processing and strand transfer in vitro, binds viral DNA sequences and promotes IN multimerisation (Engelman et al, 1993). The central catalytic core domain (CCD; residues 50–212) binds specifically to viral DNA. It contains the D, D-35E triad that coordinates divalent ions and is conserved in other retroviral integrases as well as in retrotransposon proteins such as the Tn5 transposase (Haren et al, 1999). The C-terminal domain (residues 213–288) of IN has an SH3-like fold (Eijkelenboom et al, 1999) that interacts with reverse transcriptase (Hehl et al, 2004) and binds DNA nonspecifically (Engelman et al, 1994). IN has been the focus of a large number of structural studies, and the design of a soluble IN mutant (F185K) (Jenkins et al, 1995) led to several structures of the CCD alone (Dyda et al, 1994; Maignan et al, 1998; Chiu and Davies, 2004), or with either the N- or the C-terminal domains (Chen et al, 2000; Wang et al, 2001). These structures initiated computer-aided modelling as well as negatively stained electron microscopy (EM) studies (Ren et al, 2007) that led to several models for the integration mechanism (Heuer and Brown, 1998; Gao et al, 2001; Karki et al, 2004; Wielens et al, 2005; Liao et al, 2007). In vitro integration assays showed that IN activity is increased in the presence of cellular proteins (Turlure et al, 2004). In particular, it has been shown that the transcriptional coactivator LEDGF/p75 interacts with HIV-1 IN (Cherepanov et al, 2003) as well as with other lentiviral integrases (Cherepanov, 2007) and that its expression is required for proviral integration and subsequent production of HIV-1 virions (Emiliani et al, 2005). Interaction occurs through the CCD of IN and the C-terminal integrase-binding domain (IBD) of LEGDF (Busschots et al, 2007). The function of LEDGF is to target IN to chromosomes of infected cells (Maertens et al, 2003). A structure of an IN(F185K) CCD–LEDGF IBD complex has been solved (Cherepanov et al, 2005). Unfortunately, in the absence of a structural model for the complete wild-type enzyme, none of the domain structures could give insights into the integration mechanism. To date, the poor solubility of the native full-length IN protein in vitro has hindered attempts to determine the organisation of the fully active unit. To overcome these problems, we purified a stable complex between the wild-type full-length HIV-1 IN and the human LEDGF proteins with a stoechiometry of 4 IN and 2 LEDGF as shown by mass spectrometry analysis. A cryo negatively stain EM envelope was determined, and its volume is coherent with the stoechiometry of 4 IN and 2 LEDGF molecules. Known atomic structures were fitted and refined using normal mode flexible fitting. The structure in the presence of DNA permits to assess the positions of viral and cellular DNA. The proposed model is validated by the analysis of the DNA–protein interactions found by mutational and cross-linking studies found in the literature and carried out in this study. Taken together, the data reveal the quaternary organisation of the functional integration unit highlighting the role of each of its components and lead to a model for integration of the viral DNA into the host genome. Results Purification and characterisation of the IN/LEDGF complex GST–IN and His6–LEDGF were purified separately and solubilised in 1 M NaCl in the presence of 7 mM CHAPS. The complex, which is formed upon removal of the GST tag by specific proteases and of the solubilisation agents by dialysis, was purified to homogeneity by affinity chromatography and gel filtration (Supplementary Figure 1). The stoichiometry of the partners was determined by high-mass MALDI ToF mass spectrometry analysis (Nazabal et al, 2006). Control experiments identified the mass of the two components: IN (MH+=32.6 kDa) and His6–LEDGF (MH+=63.4 kDa) (Figure 1A). In a second step, the purified complex was chemically cross-linked before mass spectrometry, which detected several protein complexes: [IN•LEDGF] (MH+=99.8 kDa), [LEDGF•LEDGF] (MH+=132.6 kDa,) [IN•2LEDGF] (MH+=165.8 kDa) but the mass of the major species corresponded to [4IN•2LEDGF] (MH+=267.4 kDa). No protein complex was detected in the higher molecular weight range between 500 and 1000 kDa. Figure 1.Functional characterisation and structure determination of the IN/LEDGF complexes. (A) IN/LEDGF complex analysis by high-mass MALDI mass spectrometry in the absence and presence of cross-linking agent showing a major peak of (IN)4–(LEDGF)2. (B) Kinetic study of the 3′ processing and strand-transfer reactions catalysed by HIV-1 integrase or by the IN–LEDGF complex using a 21- or 40-mer DNA substrate. Bands correspond to substrate DNA (S), 3′-processed product (P) and strand-transfer product (T). Incubation times are in min. The bands intensities are reported as a function of time, showing the increase of the 3′ processing kinetic when using the IN–LEDGF complex with a 40-mer DNA substrate. (C) Views of the cryo negatively stained IN/LEDGF complex (upper row) and the corresponding reprojections of the 3-D model (lower row). (D) The 3-D model of the cryo negatively stained IN/LEDGF complex. (E) The 3-D model of the cryo negatively stained IN/LEDGF complex in the presence of the 21-bp DNA substrate. Download figure Download PowerPoint Functional characterisation To investigate the 3′ processing and strand-transfer activities of the IN/LEDGF complex, we used 21- and 40-mer DNA duplexes that mimic the HIV-1 U5 viral DNA end (Figure 1B). In the presence of the 40-mer DNA, both the initial rate and the efficiency of the 3′-processing reaction are found to be higher for the IN/LEDGF complex compared with the recombinant IN alone, indicating that LEGDF stimulates the 3′-processing activity of IN. Interestingly, this increased activity is not observed when the 21-mer DNA is used, suggesting that a minimal DNA length is required to stimulate the 3′-processing reaction. Moreover, in the presence of LEGDF, the strand-transfer efficiency was strongly enhanced for both the 21- and the 40-mer DNA. Taken together, these results indicate that IN increases its biological activities upon interaction with LEDGF. Electron microscopy of the cryo negatively stained IN/LEDGF complex The IN/LEGDF complex was first observed in transmission EM after negative staining, which showed that more than 85% of the particles were homogeneous in size. The preliminary analysis of an image data set of 3732 isolated particles revealed molecular views that showed a clear two-fold symmetry either in-plane for the side view or normal to the plane for the top view. This symmetry operator was also detected in the eigenvectors before any rotational alignment, indicating that the complex contains two identical units (Supplementary Figure 2). To improve the structural preservation of the sample, a cryo negative stain method was used to observe hydrated IN–LEDGF complexes with high contrast. The particles were sandwiched between two carbon films in the presence of uranyl acetate and quick frozen before complete dehydration (Golas et al, 2005). The analysis of 11 441 molecular images again showed a two-fold symmetry and a 3-D model was calculated from 350 different views (Supplementary Figure 3A). The re-projections of the 3-D model closely matched the original views (Figure 1C). The envelope shows a triangular shape when viewed from the side (14 by 15 nm) and a highly elongated shape (10 by 15 nm) when viewed down the two-fold axis (Figure 1D). The volume enclosed by the envelope is consistent with the stoichiometry of 4 IN and 2 LEDGF molecules (∼260 kDa) determined by mass spectrometry and the resolution is of 14 Å according to the Rosenthal and Henderson criteria (Rosenthal and Henderson, 2003) (Supplementary Figure 4A). To determine the position of the IN subunit, a complex was assembled in which the IN carried a GST fusion at its N-terminus. The analysis of negatively stained particles revealed a large additional density located in the upper region of the 3-D model (Supplementary Figure 5). This experiment places IN in the upper domain of the model, a position in which the EM envelope revealed a Y-shaped clamp of 8.5 by 7 nm in size emphasised by red dots in Figure 2A. To fit the known atomic structures into the EM map, we used a composite model of the full-length IN in complex to the IBD of LEDGF (residues 347–442) obtained by the superposition of the catalytic domains, which are present in all structures (Figure 2A). This composite model could be readily placed into the Y-shaped clamp of the EM envelope. Normal modes flexible fitting in combination with rigid body refinement and structure idealisation were used to fit the integrase model into the EM map (Supplementary Figure 6, 7 and Movie 1). The fitting parameters were chosen in a way not to modify the fold of the protein domains (Supplementary Protocol 4). The C-terminal IN extensions fit into each of the branches of the 'Y', whereas the dimeric CCD is accurately positioned into the central part of the clamp. In contrast, the N-terminal domains, which fall outside of the envelope, were readily fitted into an empty region of the EM map. The final model of the complex shows no steric clashes between the different domains, and the top part of the envelope is fully occupied by the known atomic structures (Figure 2B). The remaining density forms a W-shaped structure at the bottom of the complex and is consistent with the size of an LEGDF dimer (Figure 2C). Figure 2.Docking of atomic structures into the cryoEM maps of IN/LEDGF and IN/LEDGF/DNA. (A) Composite atomic model constructed from available X-ray structures. (B) Superposition of the fitted 3-D model and the IN/LEDGF EM envelope. The Y-shaped structure that accommodates the atomic structure of the catalytic and C-terminal IN domains is contoured by red dots. (C) Surface representation of the IN/LEDGF structure: the IN tetramer is in gold and the LEDGF dimer visualised by the difference map between the atomic model and the EM map is in grey. (D) Superposition of the IN/LEDGF (gold) and IN/LEDGF/DNA (blue) envelopes. The Y-shape in the top of the envelope is highlighted with red dots. The protruding densities corresponding to DNA are circled in orange. Densities (1) and (3) are assigned to viral DNA, whereas density (2) is assigned to target DNA. (E) Atomic model fitted into the IN/LEDGF/DNA envelope. (F) Difference map between the EM map and the fitted model. Fitted DNA is shown in blue. 1 and 3 represent the viral DNA, respectively, in the integration and 3′-processing step, and 2 represents the target DNA. (G) Atomic models of IN and DNA fitted into the EM map. DNA molecules assigned to viral DNA are shown in yellow and DNA assigned to target DNA is shown in red. (H) Crystal structure of the Tn5–DNA complex superposed to the IN–CCD identifying the viral DNA in the 3′-processing position. Two viral DNA positions in the integration intermediate and 3′-processing state are represented by red and green arrows, respectively. IN is indicated in gold, the viral DNA in orange, the target DNA in red and the envelope for the LEDGF dimer in grey. Download figure Download PowerPoint Architecture of the functional IN/LEDGF/DNA complex To determine the DNA-binding sites, the IN/LEDGF complex was incubated with a 10-fold molar excess of the 21-mer U5-substrate. A data set of 11 706 images of cryo negatively stained complexes was recorded and analysed independently of the previous model. The resulting 3-D envelope was slightly more elongated than the native complex (Figure 1E). The envelopes of the IN/LEGDF and the IN/LEGDF/DNA complexes were superimposed to reveal the structural changes induced by the addition of DNA (Figure 2D). The upper domain appears larger, suggesting that additional mass is bound to the IN molecules. Comparison of the two maps revealed a displacement of the top branch of the Y domain (C-terminal domain of integrase), which results in a closure of the clamp upon DNA binding (Figure 2D). The crystal structures were adjusted and refined as described earlier (Figure 2E; Supplementary Figures 6, 8 and Movie 2). In each IN dimer, three-stain exclusion spikes are detected at the surface of the model (labelled 1, 2 and 3 in Figure 2D). These spikes are likely to reflect protruding DNA molecules that exclude the stain as shown by (Schultz et al, 1996; Wagenknecht et al, 1988) (Supplementary Figure 3B). A difference map between the EM envelope and the fitted model reveals 20 Å wide rod-like structures that are consistent with the size and shape of DNA molecules. The extremity of these rods corresponds to the above-mentioned spikes (Figure 2F; Supplementary Movie 4). Rod 1 is connected to the catalytic domains of IN, indicating that this density corresponds to oligonucleotides mimicking the viral DNA. Rod 2 lies between the two arms of the clamp and forms a continuous density with the two-fold related rod 2′ of the second clamp, thus, producing a bent filament connecting the two IN dimers. As the ends of this filament do not point towards the active sites, we interpreted this density as the target DNA. A DNA fragment from the nucleosome structure (Richmond and Davey, 2003) could be fitted into this additional volume, which is consistent with the observation that nucleosomes promote the HIV integration reaction (Pruss et al, 1994a). Moreover, the positions of the target DNA that are closest to the 3′ ends of the two viral DNA are separated by a distance of 15 Å or five base pairs, which corresponds to the documented distance of viral DNA integration into the target DNA. The structure formed by the IN/LEDGF complex and these four DNA fragments (labelled 1,1′,2,2′ in Figure 2D) could, thus, correspond to an integration intermediate between the 3′ processing and the integration step. Rod 3 and the two-fold related rod 3′ is consistent with a straight B DNA and lies at the interface with the LEDGF moiety (Figure 2D and F). To gain some insight into the role of this unexpected DNA fragment, we superimposed the atomic structure of the IN catalytic site with that of the homologous Tn5 transposase in complex with DNA (Davies et al, 2000). The path of the Tn5 DNA fits properly into rod 3, thus, indicating that the corresponding DNA fragment is in the position of the viral DNA during the 3′-processing step. The excess of 21-mer DNA can explain the concomitant occupancy of the 3′ processing and integration site of IN. Using this information, a structural model for the 3′ processing intermediate could be constructed (Figure 2H). Model validation by DNA–protein cross-linking Our proposed model fully agrees with previously published biochemical and genetic data. To further validate our structural model and to identify the protein domains in contact with the viral DNA, cross-linking experiments were carried out to identify proximal DNA bases and protein amino acids. The found cross-linked peptide had the RKAKIIRDYGK sequence corresponding to amino acids 263–273 in the C-terminal domain. This result indicates that the sixth nucleotide from the nonprocessed strand end is located near Lys264 or Lys266. Our structures are in excellent agreement with these findings, as the lysine 264 is in the vicinity of the phosphate between nucleotides 6 and 7 of the unprocessed DNA strand (Supplementary Figure 9). This confirms the importance of the C-terminal domain for the mechanism of integration and viral DNA binding. Discussion Architecture of the IN–LEDGF assembly Mass spectrometry and cryoEM showed that the complex formed between the HIV-1 integrase and its cellular cofactor LEDGF contains 4 IN and 2 LEDGF molecules. This finding is consistent with biochemical data, indicating that an IN tetramer is the minimal complex required for the concerted DNA integration reaction (Li et al, 2006). The in vitro catalytic activity and stability of this assembly are superior to that of IN molecules alone, indicating that LEGDF acts as a chaperone by preventing IN from aggregation or higher multimerisation. The existing atomic structures could be readily fitted into the EM envelope, thus, positioning the functional domains of the enzyme. Two main differences were detected between our model and the atomic structures. First, the crystal structure of the IN CCD:LEDGF IBD complex contains two copies of each protein, whereas our structure shows a 2:1 stoechiometry where each LEGDF interacts with two IN molecules. Second, according to the available X-ray crystal and NMR structures, the IN N-terminal domains form dimers with different interfaces, suggesting that this interaction is not specific (Li et al, 2006). In our model, this domain does not dimerise but instead contacts the CTD of its own IN molecule (NtA-CtA, NtB-CtB) (Figure 3A). Very recently, the structure of the two domains HIV-2 integrase (CCD and NTD) in complex with the LEDGF IBD has been solved and is in agreement with our model (Hare et al, 2009). In this structure, the IN–NTD does not dimerise and is positioned as in the EM model. Moreover, a single LEDGF molecule is associated to each IN dimer. The contact between the residues E10 from IN and R405 from LEDGF shown in the crystallographic structure is in agreement with our model, as these residues are within reach in our complex (Supplementary Figure 10). The domains arrangement has important functional consequences as the two IN molecules form a dimer, within which they adopt an asymmetric organisation and fulfil distinct functions. Thus, the 4 IN molecules (A1, A2, B1 and B2) are organised into two asymmetric IN dimers (1 and 2) related by a two-fold axis. Such an asymmetry has been inferred previously from chemical cross-linking experiments, which showed that intra-dimer and inter-dimer contacts were not equivalent, thus, excluding the possibility that the IN tetramer exhibits a four-fold symmetry (Faure et al, 2005). Figure 3.Integrase domain organisation. (A) Two perpendicular views of the (IN)4–(LEGDF)2 complex. Each IN/LEDGF complex contains 4 IN molecules (A1, A2, B1 and B2) organised in two IN dimers (1 and 2). The different IN monomers are represented with different colours: monomer A1 in light blue, A2 in dark blue, B1 in light yellow and B2 in yellow. Monomers 1 and 2 are related by a two-fold axis. The density of LEDGF (grey) is obtained by subtracting the model from the EM map. (B) Same views for the complex with DNA in the 3′-processing state. The viral DNA is shown in orange and the target DNA in red. Download figure Download PowerPoint Architecture of the IN–LEDGF–DNA complex The 3-D model of the functional IN/LEDGF/DNA assembly revealed the interaction sites and the paths of the host and viral DNA for the 3′-processing mode and for an integration intermediate. The global domain organisation of the integrase tetramer in complex with DNA is similar to the architecture of the protein assembly except for conformational changes of the N- and C-terminal parts of IN. The target DNA binds within the IN clamp. Upon DNA binding, the C-terminal domain of monomer B and the N-terminal domain of monomer A move to clamp the target DNA (Figure 3B). Our data reveal two viral DNA sites that may correspond to two distinct functional states. In the 3′-processing mode, the viral DNA ends bind integrases from two different dimers, as the DNA is positioned in the 3′-processing active site of monomer B through an interaction with monomer A from the opposite dimer. The C-terminal end of monomer A interacts strongly with both host and viral DNA, close to the integration site of the target DNA and to the 3′OH end of the viral DNA. In the integration intermediate, the viral DNA and the C-terminus of monomer A are displaced to position the 3′OH of the viral DNA towards the target integration sites separated by the canonical five base-pair stagger (15 Å) (Pruss et al, 1994b). These findings agree wit
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