Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75

526 VOLUME 12 NUMBER 6 JUNE 2005 NATURE STRUCTURAL & MOLECULAR BIOLOGY

Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75Peter Cherepanov1,2, Zhen-Yu J Sun3, Shaila Rahman1, Goedele Maertens1,2, Gerhard Wagner3 & Alan Engelman1,2

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase (IN) in human cells. We have determined the NMR structure of the integrase-binding domain (IBD) in LEDGF and identified amino acid residues essential for the interaction. The IBD is a compact right-handed bundle composed of five �-helices. Based on folding topology, the IBD is structurally related to a diverse family of �-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-�. LEDGF residues essential for the interaction with IN were localized to interhelical loop regions of the bundle structure. Interaction-defective IN mutants were previously shown to cripple replication although they retained catalytic function. The initial structure determination of a host cell factor that tightly binds to a retroviral enzyme lays the groundwork for understanding enzyme-host interactions important for viral replication.

Transcriptional coactivator p75, more commonly known as LEDGF, is a chromatin-associated protein1,2 that belongs to the hepatoma-derived growth factor (HDGF) family of proteins. Based on its copurification with transcription factor PC4, LEDGF was first proposed to play a role in transcriptional regulation3. Subsequent work revealed that LEDGF is a survival factor that protects cells from stress-induced apopto-sis (reviewed in ref. 4) and implicated LEDGF in a variety of human diseases. The t(9;11)(p22;p15) chromosomal translocation associated with acute myeloid leukemia results in the fusion of the nucleoporin NUP98 to LEDGF5. Autoantibodies to LEDGF are found in patients with atopic dermatitis and other inflammatory conditions4. In addi-tion to these ailments, LEDGF has been suggested to play a role in the replication of HIV-1, the retrovirus that causes AIDS.

The early events of HIV-1 replication proceed through a series of distinct steps. After entry into a target cell and a relatively poorly defined uncoating step, the viral RNA genome is converted into linear double-stranded cDNA by the viral enzyme reverse transcriptase in the context of the reverse transcription nucleoprotein complex (reviewed in ref. 6). After nuclear import, IN catalyzes the insertion of the viral cDNA into a cell chromosome7. An essential step in the retroviral life cycle, integra-tion also occurs in the context of a higher-order nucleoprotein complex, the preintegration complex (PIC). In addition to IN, the PIC contains a variety of host cell proteins that are likely to play cofactor or auxiliary roles in integration8.

LEDGF is the dominant binding partner of HIV-1 IN in human cells2,8–12. When expressed in the absence of other viral proteins, HIV-1 IN accumulates in the nucleus13,14 and stably associates with chroma-tin14,15. Short interfering RNA-mediated knockdown of endogenous

LEDGF or overexpression of a nuclear localization-defective mutant of LEDGF resulted in major intracellular redistribution of HIV-1 IN9,10,16. Most notably, upon LEDGF depletion, the viral protein lost its ability to associate with chromatin9,10, implying that LEDGF is the main chromatin-tethering factor for IN. HIV-1 IN is inherently unstable as it is a substrate for ubiquitination and degradation by the proteasome15,17, and the interaction with LEDGF protected IN from this fate9,11. In in vitro assays, LEDGF potently stimulated the integra-tion activity of HIV-1 IN2,18. LEDGF interacts with other lentiviral INs (HIV-2 (ref. 19) and feline immunodeficiency virus (FIV)10), but not with oncoretroviral INs10,19. In accordance with a potential role in lentiviral replication, LEDGF was found in HIV-1 and FIV PICs10. One hypothesized function for LEDGF is that of a chromatin-tethering factor for PICs during integration20,21.

HIV-1 IN is composed of three functional domains (reviewed in ref. 7) and studies conducted in human cells revealed that the catalytic core domain (CCD) is both necessary and sufficient for the interaction with LEDGF9. The IBD in LEDGF, initially localized within a 205-residue C-terminal region of the protein9, was subsequently defined as an ∼80-residue evolutionarily conserved domain that resisted proteolysis18. The IBD is essential for LEDGF-mediated stimulation of HIV-1 integra-tion18. The IBD and the N-terminal PWWP domain (named after a signature Pro-Trp-Trp-Pro motif) constitute the most conserved regions of LEDGF. Whereas the PWWP domain is found in a variety of chromatin-associated proteins including the five members of the HDGF family, the IBD is restricted to LEDGF and HDGF-related protein 2 (HRP2)18. Notably, HRP2 also binds to and stimulates HIV-1 IN activity18. The identification of the IBD as a distinct LEDGF subdomain, as well as its

1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA. 2Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA. Correspondence should be addressed to A.E. ([email protected]).

Published online 15 May 2005; doi:10.1038/nsmb937

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NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 12 NUMBER 6 JUNE 2005 527

conservation within HRP2, has been recently confirmed using comple-mentary methods22. Unlike LEDGF, HRP2 seems to lack the ability to tether IN to chromatin22.

The initial structural determination of a PWWP domain was that of DNA methyltransferase Dnmt3b, solved by X-ray crystallography23. A subsequent NMR study revealed a similar fold for the PWWP domain in human HDGF24. To understand the structural basis for the recognition between LEDGF and HIV-1 IN, we determined the solution structure of the IBD. This afforded an extensive structure-based mutagenesis, the results of which identified key LEDGF residues essential for interac-tion with IN. In addition, we analyzed several HIV-1 IN CCD mutants with established viral replication phenotypes for binding to LEDGF, and based on these results proposed the location of the LEDGF-binding site on IN. Our study reveals the structure of the second conserved domain in LEDGF, a chromatin-associated factor involved in a variety of human disorders.

RESULTSSolution structure of the LEDGF IBDAlthough residues 347–429 of LEDGF were sufficient for binding to HIV-1 IN18,22, the somewhat larger LEDGF(347–471) fragment was chosen for NMR studies owing to higher yields and favorable solubility upon expression in bacteria. Based on results of analytical ultracentrifuga-

tion experiments (Supplementary Methods online), full-length LEDGF protein was monomeric in solution (Supplementary Fig. 1 online). As we expected, sedimentation equilibrium data for LEDGF(347–471) also fitted to a monomer, up to the highest tested concentration (0.35 mM) (data not shown). Using multidimensional NMR spectroscopy, we obtained assignments of 1H, 13C and 15N resonances for residues 347–471 of LEDGF. According to NOESY spectra, residues 348–428 represented the structured region in this fragment. Scarcity of NOEs observed for residues C-terminal to Gly430, poor chemical shift disper-sion on the 2D 1H-15N HSQC spectrum, and fast hydrogen-deuterium exchange of the main chain amide protons collectively suggested that residues 430–471 were not involved in a stable fold. Structures were calculated with 1,034 NOE restrains (Table 1). For the final ensemble of 15 structures, the r.m.s. deviation of backbone atoms from the mean structure was 0.29 Å (Supplementary Fig. 2 online).

The IBD forms a compact right-handed helical bundle, which is composed of four main α-helices: α1, α2, α4 and α5. Two hairpins formed by helices α1-α2 and α4-α5 are connected by a shorter helix, α3, that crosses the bundle at the bottom of the structure (Fig. 1a,b; the terms ‘top’ and ‘bottom,’ used throughout the text, refer to the orientation in Fig. 1a). Residues 364–367, located in the loop region of the first hairpin, form one turn of a 310-helix. The bundle adopts a left-handed twist almost invariably observed in natural coiled coils

Figure 1 NMR structure of the LEDGF IBD. (a) Stereo view of a ribbon diagram (residues 348–428). Side chains of all hydrophobic residues (leucine, isoleucine, valine, methionine, cysteine, phenylalanine and tyrosine) and Asp366 are shown as sticks. Ile365, Asp366 and Phe406, which mediate binding to HIV-1 IN (see below), are highlighted in red. Methionine and cysteine residues are yellow. (b) Sequence alignment of IBD homology regions from several LEDGF and HRP2 orthologs. Invariant residues are white on red background; residues with conserved properties are bold on yellow background. GenBank accession numbers for LEDGF sequences: human, AAC25167; mouse, AJ308965; lizard, CF775831; chicken, AY728140; frog, AY728141. HRP2 sequences: human, NP_116020; mouse, NP_032259; fish, AY728142. The HRP2 frog sequence is from ref. 18. Residue numbering shown above the alignment corresponds to the sequence of human LEDGF. Positions of alanine-scanning mutations are indicated with triangles (blue, mutations with no or little affect on IN binding; red, mutations that ablated IN binding; orange, substantially reduced binding) above the alignment (see Fig. 2 below). Residue solvent accessibility (white, buried; light blue, partially accessible; dark blue, exposed) and secondary structure (α-helices α1, α2, α3, α4 and α5, determined by PROCHECK-NMR52, are indicated below the alignment. (c) Surface electrostatic potential of the LEDGF IBD (blue, positive; red, negative). Left, the structure is oriented as in a; right, produced by a 180° rotation around the vertical axis. Side chains of selected exposed residues are indicated.

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and helix bundles. However, the topology of the IBD is dissimilar to classical four-helix bundles that feature antiparallel arrangements of neighboring helices, such as those found in cytochromes, human growth hormone and ferritin25.

A search for structural homologs using the Dali server (http://www.ebi.ac.uk/dali/) resulted in ten hits with Z-scores between 8.7 and 7.0, all corresponding to HEAT (named after Huntingin, elongation fac-tor 3, subunit A of phosphatase 2A, yeast PI3 kinase TOR) or ARM (named after Drosophila melanogaster protein Armadillo) repeats of various proteins. Structurally and evolutionarily related HEAT and ARM repeats mediate protein-protein interactions that are involved in a wide variety of cellular processes26. Individual ARM and HEAT repeats are composed on average of 40 residues and fold into bihelical hairpins. In canonical HEAT/ARM repeat proteins, tandem repeats stack together to form continuous superhelical domains. According to the Dali scores, the IBD showed high structural similarity to HEAT repeats of eukaryotic initiation factor eIF4G27 and karyopherin-β28 (Supplementary Fig. 3 online). Although it has a similar fold, the IBD does not have recog-nizable sequence homology to HEAT or ARM repeats. The IBD does not seem to be a unique example of a HEAT-like topology in a small helical domain. A similar fold was described for the pseudo HEAT repeat analogous topology (PHAT) domain of D. melanogaster RNA-binding protein Smaug29 (Supplementary Fig. 3) and the HEAT analogous motif (HAM) domain of the small subunit of the eukaryotic initiation factor eIF3 that features two helical hairpins30.

Most nonpolar residues are buried in the interior of the IBD struc-ture, forming an extensive hydrophobic core (Fig. 1a,b). Several exposed or partially exposed hydrophobic residues (Ile365, Leu368, Phe406 and Val408) belonging to loop regions cluster at the top of the structure. As shown below, Ile365, and probably Phe406 and Val408, are involved in the interaction with HIV-1 IN. Thus, a relatively hydrophobic surface at the top of the IBD structure is likely the principal protein-binding site. The charge distribution is not uniform over the surface of the molecule, as several positively charged residues cluster to one side (Fig. 1c). The majority of these residues (Lys360, Lys401, Lys402, Arg404, Arg405, Lys407 and Lys424) are conserved in the LEDGF and HRP2 IBDs (Fig. 1b), suggesting functional importance. We have not observed any appreciable affinity of the LEDGF IBD for DNA in vitro (data not shown), and neither the sequence nor the structural fold shows similarity to known DNA- or RNA-

binding domains. We speculate that the IBD might interact with proteins carrying complementarily negatively charged residues, or, in the context of the full-length protein, form intramolecular salt bridges.

The IN-binding interface of LEDGFTo identify LEDGF residues involved in the interaction with HIV-1 IN, the IBD was studied by alanine-scanning mutagenesis. Mutations were guided by the alignment of LEDGF and HRP2 orthologs and by the NMR structure. In total, 26 amino acids were targeted, including almost all exposed or partially exposed hydrophobic residues (Fig. 1b). The mutants, in the context of LEDGF(347–471), were expressed in bacte-ria, purified and tested for binding to IN in a His6-tag pull-down assay. Alteration of LEDGF residues Ile365, Asp366 or Phe406 abrogated bind-ing to IN (Fig. 2a). Because the D366N mutant also failed to bind IN, the negative charge at this position is essential for the interaction. Ile365 and Asp366 are located within the 310-helical region of the loop between α1 and α2 (Fig. 1b) and are unlikely to be essential for overall stability of the domain. Accordingly, the I365A, D366N, and F406A IBD mutant proteins retained high expression levels and solubility. The IBD was first recognized as a trypsin-resistant domain of full-length LEDGF18. The mutant IBD proteins showed wild-type resistance to proteolysis, further arguing that the mutations did not disrupt the native fold (data not shown). Of note, isoleucine and aspartate residues are enriched among protein interaction hot spots31, supporting their direct involve-ment in the interaction with IN. We are cautious, however, to conclude that Phe406 makes a direct contact with IN, because it is only partially exposed in the structure and is involved in multiple hydrophobic inter-actions with loop and terminal α-helical residues, and thus may be important for the proper organization or stability of the hairpin loops. HRP2 harbors aspartate (residue 489) at the position corresponding to Asp366 in LEDGF (Fig. 1b). However, valine and tyrosine substitute for Ile365 and Phe406 in LEDGF, respectively. We speculate that these changes are likely to explain the apparent reduced affinity of HRP2 for IN18,22. Among the other tested LEDGF mutants, substitution of Val408 or Lys415 substantially reduced binding. However, K415A was the least soluble of 27 tested mutants, arguing that the observed effect on binding might be indirect. Notably, all mutations that abolished the binding of LEDGF to IN mapped to the top of the IBD structure (Fig. 2b), strongly implying that this is the IN interaction interface.

Figure 2 Mapping the IN-binding interface. (a) His6-tag pull-down assay. LEDGF(347–471) or its point mutants were incubated with His6-tagged HIV-1 IN and Ni-NTA agarose beads. IN was omitted from the sample in the leftmost lane. Proteins bound to beads were separated by tricine 10–20% w/v PAGE and detected by staining with Coomassie blue. Seventy-five percent of the input quantity of each LEDGF protein, representing the amount expected to bind to IN considering a 1:1 stoichiometry, is shown at the bottom. (b) Surface map of the LEDGF IBD painted according to the results of the pull-down assay: red, side chains of residues that when mutated abolished IN binding (Ile365, Asp366 and Phe406); pink, residues (Val408 and Lys415) that when mutated gave an intermediate phenotype; blue, residues that did not substantially contribute to binding. The rest of the structure is gray. Left-hand view is oriented as in Figure 1a; middle, 180° rotation around the vertical axis; right, view from top of the structure.

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preintegration blocks34–37. Because these INs retained enzyme activity, the block in virus replication might be explained by the inability of the mutant proteins to interact with viral and/or host cofactor(s)34–37. A reduced affinity of HIV-1 IN mutant V165A for LEDGF was previously documented8. This mutant failed to coimmunoprecipitate detectable amounts of endogenous LEDGF from human cell extracts. Furthermore, the steady-state level of V165A IN was substantially lower than that of the wild-type protein, suggestive of rapid turnover of the mutant protein.

We have expanded pull-down analyses to include several class I and class II IN CCD mutant proteins. Class II mutants V165A, R166A, and L172A K173A were most defective for binding, as each was unable to recover detectable amounts of LEDGF in the His6-tag pull-down assay (Fig. 4, lanes 6–8). In addition, these mutants failed to interact with GST-LEDGF or GST fused to the HRP2 IBD in GST pull-down experi-ments (data not shown). In sharp contrast, class I IN mutants Q62K and D64N were able to bind LEDGF, albeit with somewhat reduced affinity as compared with wild-type IN (lanes 3–5). A separate class II IN mutant protein, F185K, derived from a virus that exhibited severe late stage defects37,38, also recovered detectable levels of LEDGF (Fig. 4, lane 9). Our data support the previous report that the CCD of IN contains the primary LEDGF-binding interface9 and suggest that it is located in the vicinity of IN residues 165–173.

DISCUSSIONThe LEDGF IBD is structurally homologous to a pair of HEAT repeats, and can be classified as a PHAT domain similar to the PHAT domain in Smaug29. The hairpin loop regions of the IBD partake in binding to HIV-1 IN and probably represent the general protein-binding interface of the domain. Given a role for LEDGF in transcriptional regulation3,39, it seems likely that components of transcriptional machinery are the natural interactors of the LEDGF IBD.

It seems that binding to LEDGF is conserved among the Lentiviridae subfamily of retroviruses and has so far been demonstrated for HIV-1, HIV-2 and FIV INs2,8,10,19. In contrast, two other well studied IN-binding partners, integrase-interactor 1 and uracil DNA glycosylase 2 (UNG2), seem HIV-1-specific40,41. It remains to be established what role LEDGF and/or its close homolog HRP2 play in lentiviral replica-tion. Among several possibilities are protection of lentiviral PICs from degradation42 and their targeting to actively transcribed genes during integration. Surprising differences in integration site selection by onco-viruses versus lentiviruses have suggested the presence of different chro-

When introduced into full-length LEDGF, the D366N mutation abolished the ability of IN to bind LEDGF in vitro (Fig. 3a). The D366N mutation also abolished the interaction in live cells: FLAG-tagged IN was effi-ciently recovered through coimmunoprecipitation with HA-tagged wild-type LEDGF but not with the mutant protein or cyclophilin A, which served as a negative control (Fig. 3b, compare lanes 5–8). The steady-state levels of IN-FLAG in cells coexpressing LEDGF D366N were similar to those observed in cells coexpressing cyclophilin A, and considerably lower than in cells overexpressing wild-type LEDGF (lanes 1–4, anti-FLAG). This observation corroborates previous reports that the interaction with LEDGF increased the steady-state levels of IN in human cells9,11 and confirms the inability of LEDGF D366N to interact with IN in live cells. Furthermore, compared with the wild-type protein, the D366N mutant did not stimulate IN activity in an in vitro integration assay (Fig. 3c). As previously reported18, 0.2 µM LEDGF substantially stimulated the ability of IN to promote the integration of a 4.7-kb mini-HIV DNA substrate32, converting a considerable fraction of the substrate DNA into various integration products (Fig. 3c, lane 5). Only negligible strand transfer activity was observed in the absence of LEDGF (lane 2) or in the presence of LEDGF D366N (lanes 8–11). Taken together, these results demonstrate that the D366N mutation abolished the IN-LEDGF interaction both in vitro and in vivo and confirm that a direct interaction with IN is required for stimulation of HIV-1 integration in vitro18.

LEDGF-binding site on INThe genetics of retroviral integration is fairly complex33. IN mutations have been divided into two classes, according to the types of viral rep-lication defects they cause. Class I IN mutant viruses are specifically blocked at integration, and often correlate with a loss of enzyme activ-ity. Active site mutants, such as D64N, present a paradigm of this class. Less understood class II mutant viruses are also blocked at integration but in addition present defects at late (assembly and release) and/or early (reverse transcription) stages of virus replication. The majority of rep-lication-defective HIV-1 IN mutant viruses fall into class II and a subset of these, which include V165A, R166A, and L172A K173A, cause potent

Figure 3 Asp366 is essential for the binding of full-length LEDGF to IN and for stimulation of IN activity. (a) His6-tag pull-down assay. Lanes 1 and 2 contained input quantities of wild-type (WT) and mutant LEDGF, respectively. Lanes 3–6, proteins bound to Ni-NTA agarose beads after incubation with His6-tagged IN (lanes 5 and 6) and/or wild-type (lanes 3 and 5) or D366N (lanes 4 and 6) LEDGF. Proteins were separated by 4–20% (w/v) SDS-PAGE and detected by Coomassie blue staining. (b) Extracts of HeLa cells (lanes 1–4) expressing IN-FLAG along with HA-tagged cyclophilin A (CypA-HA, lanes 1 and 5), LEDGF (lanes 2 and 6), HA-LEDGF (lanes 3 and 7), or HA-LEDGF D366N (lanes 4 and 8). Proteins recovered after immunoprecipitation with an HA affinity matrix (lanes 5–8) were analyzed by western blotting (WB) using mouse anti-HA, anti-LEDGF, anti-FLAG or anti-β-actin as indicated. Migration positions of molecular mass standards (kDa) are shown alongside the gel in the top panel. (c) Strand transfer activity of HIV-1 IN in the presence of WT or mutant LEDGF. Mini-HIV DNA substrate32 was incubated in the presence (+) or absence (–) of HIV-1 IN and/or LEDGF (wild type, lanes 4–7; D366N, lanes 8–11). Reaction products separated by agarose gel electrophoresis were visualized by staining with ethidium bromide. Migration positions of the substrate DNA, strand transfer products, and DNA markers (kb) are indicated.

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matin targeting factors for these subfamilies of retroviruses (reviewed in refs. 20,21). The specificity of LEDGF for lentiviral INs together with its ability to tether IN to chromatin argues in favor of LEDGF as being such a targeting factor. Furthermore, the IN proteins derived from replica-tion-defective mutant viruses V165A, R166A, and L172A K173A failed to interact with LEDGF. Because these INs were enzymatically active yet the viruses experienced potent preintegration blocks34–37, we tentatively speculate that LEDGF and/or its homolog HRP2 may be essential for lentiviral replication. However, the pleiotropic nature of class II IN muta-tions calls for a note of caution. The hallmark of the class II phenotype is a reduction in the accumulation of late reverse transcription prod-ucts33,37. We cannot rule out the possibility that mutations within the 165–173 region of IN lead to improper PIC assembly, resulting in insta-bility of the nucleoprotein complex. In addition, Leu172 was implicated in binding to UNG2 (refs. 36,43). Although UNG2 is more likely to play a role during reverse transcription than integration41, it seems possible that multiple IN-host protein interactions could be disrupted by these mutations. It is clear that more work is needed to ascertain the role(s) of these cellular proteins in viral replication. Further analyses of class II IN mutant viruses and/or the generation of LEDGF/HRP2 genetic knockout cell lines might shed light on these questions.

Our study unveiled the first structure of a high-affinity interactor of a retroviral enzyme, setting a precedent for understanding viral enzyme-host interactions important for replication and laying the groundwork for further research into the cellular function(s) of LEDGF. Given intense interest in developing retroviral and lentiviral vectors as well as their potential drawbacks44, we speculate that further structural insights into the IN-LEDGF interaction will prove useful for modulating the biological properties of this class of DNA recombinases.

METHODSDNA constructs. Plasmids pCP-GST75-81, pKB-IN6H and pCP-Nat75, used for bacterial expression of GST-tagged LEDGF(347–471), HIV-1 IN with a C-termi-nal His6-tag, and nontagged human LEDGF, respectively, have been described9,18. To make pGM-P75, which was used for expression of nontagged LEDGF in human cells, an EcoRI-BamHI fragment of pCP6H75 (ref. 2) containing the entire LEDGF open reading frame was subcloned between EcoRI and XhoI sites of pGM-SV40NLS-LacZ16. To allow ligation, BamHI and XhoI termini were filled in using Pfu DNA polymerase (Stratagene). Plasmids used for expression of cyclophilin A carrying a C-terminal HA tag, FLAG-tagged HIV-1 IN (pCP-INsalaFLAG), and HA-tagged LEDGF (pBHA-P75), have been described2,18,45.

Point mutations were introduced into pCP-GST75-81, pKB-IN6H, pCP-Nat75 and pBHA-P75 using QuikChange mutagenesis (Stratagene) and primers designed according to the manufacturer’s recommendations.

Recombinant proteins. His6-tagged IN and non-tagged full-length LEDGF were purified as described9. To obtain LEDGF(347–471) for NMR experiments, BL21 Escherichia coli cells transformed with pCP-GST75-81 and grown to an A600 of 0.9–1.0 in 6 l of LB or minimal salt medium containing 2 g l–1 13C-substi-tuted glucose and/or 1 g l–1 15NH4Cl for uniform labeling (Cambridge Isotope Laboratories) were induced with 0.4 mM IPTG at 28 °C for 4 h. The cells were lysed by sonication in 500 mM NaCl, 1 mM EDTA, 0.5 mM PMSF, 5 mM DTT. The lysate was precleared by centrifugation and incubated with 8 ml of gluta-thione Sepharose gel (Amersham-Pharmacia) for 4 h at 4 °C. After washing in an excess of 200 mM NaCl, 50 mM Tris-HCl, pH 7.4, the gel (50% (v/v) suspension) was incubated with 100 units of human thrombin (Sigma-Aldrich). After 3 h at 25 °C, the supernatant was diluted with four volumes of 50 mM sodium phosphate, pH 7.2, and injected into a 5-ml SP Sepharose column (Amersham-Pharmacia). The protein was eluted with a linear 0–0.5 M gradient of NaCl in 50 mM sodium phosphate, pH 7.2, further purified by gel filtration on a Superdex-200 column in 100 mM NaCl, 50 mM sodium phosphate, pH 7.25, and concentrated using a Centriprep YM-3 device (Millipore).

NMR spectroscopy and structure determination. LEDGF(347–471) with GST tag–derived glycine-serine at its N terminus was used in all NMR experi-ments. Spectra (0.5-0.75 mM protein in 100 mM NaCl, 50 mM sodium phos-phate, pH 7.25, 90% H20, 10% D2O) were recorded at 25 °C on Bruker 600, Bruker 500 and Varian 600 spectrometers equipped with cryogenic probes, or on a Bruker 750 spectrometer with a room-temperature probe. For 2D-NOESY, 13C-NOESY, HCCH-TOCSY, and a 15N-HSQC spectrum for hydrogen-deuterium exchange, the buffer was replaced with 100% D2O containing 100 mM NaCl, 38 mM Na2HPO4 and 12 mM NaH2PO4. Sequential assignments were obtained using IBIS46 on the basis of pairs of standard triple-resonance experiments HNCA/HN(CO)CA, HN(CA)CB/HN(CO)(CA)CB and HNCO/HN(CA)CO, recorded on uniformly 15N- and 13C-labeled protein47,48. Side chain proton reso-nances were assigned on the basis of triple resonance experiments C(CO)NH, H(C)(CO)NH and HCCH-TOCSY. Stereospecific assignment of methyl groups of leucine and valine residues was obtained from 13C-HSQC spectrum of 10% 13C-labeled protein49.

Torsion angles ϕ and ψ were predicted from 1Hα, 13Cα, 13Cβ, C′ and backbone 15N chemical shifts using TALOS50. Structures were calculated using simulated annealing and refinement protocols of X-PLOR version 3.851 (ref. 51) based on a total of 1,093 (1,043 for the structured region) experimental NOE restraints and 150 dihedral angle restraints. Fifty-six additional restraints based on 28 hydrogen bonds confirmed by both 15N-NOESY and the hydrogen-deuterium exchange experi-ment were used to reduce the r.m.s. deviation of the final ensemble of structures (Table 1 and Supplementary Fig. 2). Structures were analyzed with AQUA and PROCHECK-NMR52. For the final ensemble of 15 structures, 88.8% of the residues were in the core and 11.2% in the additionally allowed regions of the Ramachandran plot. The molecular structure figures were generated using MolMol53.

His6-tag pull-down assay. Samples containing 20 µl Ni2+-nitrilotriacetic acid (Ni-NTA) agarose beads (settled bead volume) (Qiagen), 10 µg BSA, 6 µg His6-tagged IN, 3 µg LEDGF(347–471), or 6 µg full-length LEDGF or their mutant forms in 400 µl pull-down buffer (PB, 150 mM NaCl, 2 mM MgCl2, 25 mM imidazole, 0.1% (v/v) Nonidet P40, 25 mM Tris-HCl, pH 7.4) were rocked for 4–5 h at 4 °C. The beads collected by gentle centrifugation were washed thrice with 600 µl of cold PB. Bound proteins eluted in 16 µl of SDS-PAGE sample buffer supplemented with 200 mM imidazole and 100 mM DTT were separated on Tris-glycine or tricine gels. Where indicated, IN was omitted to control for nonspecific binding of LEDGF to Ni-NTA agarose beads.

Cell transfection and immunoprecipitation. HeLa cells were cultured in DMEM supplemented with 10% (v/v) FBS. Cells grown to 80% confluency in six-well dishes were transfected with 3 µg of plasmid expressing CypA-HA, LEDGF, HA-LEDGF or HA-LEDGF D366N along with 1 µg of IN-FLAG expression vector using Lipofectamine-2000 (Invitrogen). Cells were lysed 36 h after transfection

Figure 4 Effect of IN mutations on binding to LEDGF in vitro. Lane 1, input LEDGF; lane 2, mock pull-down without IN; lanes 3–9, wild-type (WT) His6-tagged IN or the indicated mutant was incubated with LEDGF and Ni-NTA agarose beads. Proteins bound to agarose beads were analyzed by SDS-PAGE and detected by staining with Coomassie. The migration positions of molecular mass markers (kDa) are indicated.

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in 150 µl 400 mM NaCl, 1 mM MgCl2, 10% (w/v) sucrose, 0.5 mM DTT, 10 mM PIPES, pH 6.8, 0.5% (v/v) NP-40 in the presence of EDTA-free complete protease inhibitor cocktail (Roche). For immunoprecipitation, samples (120 µl) precleared with 15 µl of protein A Sepharose beads were incubated with 12 µl HA.11 affin-ity matrix (Covance). After 3-h rocking at 4 °C, the beads were washed once in extraction buffer followed by three changes of 100 mM NaCl, 1 mM MgCl2, 10% (w/v) sucrose, 0.5 mM DTT, 10 mM PIPES, pH 6.8, 0.1% (v/v) NP-40. Bound proteins were eluted in 60 µl of SDS-PAGE sample buffer. Cell extracts and immunoprecipitates were analyzed by western blotting using monoclonal anti-HA 16B12 (Covance), anti-LEDGF (BD Biosciences), anti-FLAG M2 or anti-β actin (Sigma-Aldrich).

Integration assay. Linearized double-stranded, blunt-ended DNA carrying ter-minal HIV-1 sequences (mini-HIV32) was preincubated with 0.6 µM IN at room temperature for 7 min. Wild-type or D366N LEDGF was then added at the indi-cated concentrations, and the samples were incubated at 37 °C for 100 min. Final reactions (20 µl) contained 150 ng substrate DNA, 110 mM NaCl, 5 mM MgCl2, 10 mM DTT, 4 µM ZnCl2, and 10 mM HEPES, pH 7.5. Reactions were stopped by addition of 25 mM EDTA, 0.5% (w/v) SDS and deproteinized by incubation with 10 µg proteinase K at 50 °C for 30 min. After ethanol precipitation, DNA products were separated in a 0.75% (w/v) agarose gel and visualized by staining with ethidium bromide.

Accession codes. The atomic coordinates for the ensemble of 15 NMR structures of the LEDGF IBD have been deposited in the Protein Data Bank (accession code 1Z9E). BIND identifier (http://bind.ca): 262757.

Note: Supplementary information is available on the Nature Structural & Molecular Biology website.

ACKNOWLEDGMENTSWe thank N. Vandegraaff for thoughtful discussions and comments on the manuscript and P. Dormitzer for stimulating discussion in the early phase of the project. We also thank J. Miranda, J. Al-Bassam and S. Harrison for the use of and help with the analytical ultracentrifuge. This work was supported by US National Institutes of Health grants GM47467 and AI37581 (G.W.), and AI39394 and AI62249 (A.E.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 14 March; accepted 13 April 2005Published online at http://www.nature.com/nsmb/

1. Nishizawa, Y., Usukura, J., Singh, D.P., Chylack, L.T., Jr. & Shinohara, T. Spatial and temporal dynamics of two alternatively spliced regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and p52, in the nucleus. Cell Tissue Res. 305, 107–114 (2001).

2. Cherepanov, P. et al. HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J. Biol. Chem. 278, 372–381 (2003).

3. Ge, H., Si, Y. & Roeder, R.G. Isolation of cDNAs encoding novel transcription coactiva-tors p52 and p75 reveals an alternate regulatory mechanism of transcriptional activa-tion. EMBO J. 17, 6723–6729 (1998).

4. Ganapathy, V., Daniels, T. & Casiano, C.A. LEDGF/p75: a novel nuclear autoantigen at the crossroads of cell survival and apoptosis. Autoimmun. Rev. 2, 290–297 (2003).

5. Morerio, C. et al. t(9;11)(p22;p15) with NUP98-LEDGF fusion gene in pediatric acute myeloid leukemia. Leuk. Res. 29, 467–470 (2005).

6. Goff, S.P. Genetic control of retrovirus susceptibility in mammalian cells. Annu. Rev. Genet. 38, 61–85 (2004).

7. Craigie, R. Retroviral DNA integration. In Mobile DNA II (eds. Craig, N.L., Craigie, R., Gellert, M. & Lambowitz, A.M.) 613–630 (ASM Press, Washington, DC, 2002).

8. Turlure, F., Devroe, E., Silver, P.A. & Engelman, A. Human cell proteins and human immunodeficiency virus DNA integration. Front. Biosci. 9, 3187–3208 (2004).

9. Maertens, G. et al. LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J. Biol. Chem. 278, 33528–33539 (2003).

10. Llano, M. et al. LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes. J. Virol. 78, 9524–9537 (2004).

11. Llano, M., Delgado, S., Vanegas, M. & Poeschla, E.M. LEDGF/p75 prevents proteasomal degradation of HIV-1 integrase. J. Biol. Chem. 279, 55570–55577 (2004).

12. Maertens, G., Vercammen, J., Debyser, Z. & Engelborghs, Y. Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75. FASEB J. Epub ahead of print, 23 March 2005 (doi:10.1096/fj.04–3373fje).

13. Pluymers, W., Cherepanov, P., Schols, D., De Clercq, E. & Debyser, Z. Nuclear localiza-tion of human immunodeficiency virus type 1 integrase expressed as a fusion protein with green fluorescent protein. Virology 258, 327–332 (1999).

14. Cherepanov, P. et al. High-level expression of active HIV-1 integrase from a synthetic gene in human cells. FASEB J. 14, 1389–1399 (2000).

15. Devroe, E., Engelman, A. & Silver, P.A. Intracellular transport of human immunodefi-ciency virus type 1 integrase. J. Cell Sci. 116, 4401–4408 (2003).

16. Maertens, G., Cherepanov, P., Debyser, Z., Engelborghs, Y. & Engelman, A. Identification and characterization of a functional nuclear localization signal in the HIV-1 integrase interactor LEDGF/p75. J. Biol. Chem. 279, 33421–33429 (2004).

17. Mulder, L.C. & Muesing, M.A. Degradation of HIV-1 integrase by the N-end rule path-way. J. Biol. Chem. 275, 29749–29753 (2000).

18. Cherepanov, P., Devroe, E., Silver, P.A. & Engelman, A. Identification of an evolu-tionarily conserved domain in human lens epithelium-derived growth factor/transcrip-tional co-activator p75 (LEDGF/p75) that binds HIV-1 integrase. J. Biol. Chem. 279, 48883–48892 (2004).

19. Busschots, K. et al. The interaction of LEDGF/p75 with integrase is lentiviral-spe-cific and promotes DNA binding. J. Biol. Chem. Epub ahead of print, 4 March 2005 (doi:10.1074/jbc.M411681200).

20. Bushman, F.D. Targeting survival: integration site selection by retroviruses and LTR-retrotransposons. Cell 115, 135–138 (2003).

21. Engelman, A. The ups and downs of gene expression and retroviral DNA integration. Proc. Natl. Acad. Sci. USA 102, 1275–1276 (2005).

22. Vanegas, M. et al. Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering. J. Cell Sci. 118, 1733–1743 (2005).

23. Qiu, C., Sawada, K., Zhang, X. & Cheng, X. The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds. Nat. Struct. Biol. 9, 217–224 (2002).

24. Sue, S.C., Chen, J.Y., Lee, S.C., Wu, W.G. & Huang, T.H. Solution structure and heparin interaction of human hepatoma-derived growth factor. J. Mol. Biol. 343, 1365–1377 (2004).

25. Kamtekar, S. & Hecht, M.H. Protein Motifs. 7. The four-helix bundle: what determines a fold? FASEB J. 9, 1013–1022 (1995).

Table 1 Structural statistics for 15 final NMR structuresLEDGF(348–428)

NMR distance and dihedral constraints

Distance constraints

Total NOE 1,034

Intra-residue 278

Inter-residue 756

Sequential (|i – j| = 1) 255

Medium-range (1< |i – j| ≤ 4) 262

Long-range (|i – j| > 4) 239

Hydrogen bonds 28

Total dihedral angle restraints

φ 67

ψ 67

Structure statisticsa

Violations (mean and s.d.)

Distance constraints (Å) 0.024 ± 0.001

Dihedral angle constraints (°) 0.23 ± 0.02

Max. distance constraint violation (Å) 0.295

Max. dihedral angle violation (°) 2.9

Deviations from idealized geometry

Bond lengths (Å) 0.00188 ± 0.00005

Bond angles (°) 0.500 ± 0.04

Impropers (°) 0.39 ± 0.03

R.m.s. deviation from mean structure (Å)

Heavy 0.86

Backbone 0.29

Data are for the structured region, residues 348–428. The final 15 structures were selected from 20 models initially calculated using X-PLOR51 based on lowest NOE energy function, no violations of NOE restraints by >0.3 Å, or dihedral restraints by >5°.aAll 15 structures from the final ensemble were included in the analysis.

A R T I C L E S©

2005

Nat

ure

Pub

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ng G

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ww

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atur

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smb


26. Andrade, M.A., Petosa, C., O’Donoghue, S.I., Muller, C.W. & Bork, P. Comparison of ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18 (2001).

27. Marcotrigiano, J. et al. A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery. Mol. Cell 7, 193–203 (2001).

28. Chook, Y.M. & Blobel, G. Structure of the nuclear transport complex karyopherin-β2-Ran x GppNHp. Nature 399, 230–237 (1999).

29. Green, J.B., Gardner, C.D., Wharton, R.P. & Aggarwal, A.K. RNA recognition via the SAM domain of Smaug. Mol. Cell 11, 1537–1548 (2003).

30. Wei, Z. et al. Crystal structure of human eIF3k, the first structure of eIF3 subunits. J. Biol. Chem. 279, 34983–34990 (2004).

31. Bogan, A.A. & Thorn, K.S. Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1–9 (1998).

32. Cherepanov, P. et al. Activity of recombinant HIV-1 integrase on mini-HIV DNA. Nucleic Acids Res. 27, 2202–2210 (1999).

33. Engelman, A. In vivo analysis of retroviral integrase structure and function. Adv. Virus Res. 52, 411–426 (1999).

34. Bouyac-Bertoia, M. et al. HIV-1 infection requires a functional integrase NLS. Mol. Cell 7, 1025–1035 (2001).

35. Dvorin, J.D. et al. Reassessment of the roles of integrase and the central DNA flap in human immunodeficiency virus type 1 nuclear import. J. Virol. 76, 12087–12096 (2002).

36. Priet, S., Navarro, J.M., Querat, G. & Sire, J. Reversion of the lethal phenotype of an HIV-1 integrase mutant virus by overexpression of the same integrase mutant protein. J. Biol. Chem. 278, 20724–20730 (2003).

37. Lu, R. et al. Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication. J. Virol. 78, 12735–12746 (2004).

38. Jenkins, T.M., Engelman, A., Ghirlando, R. & Craigie, R. A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in mul-timerization. J. Biol. Chem. 271, 7712–7718 (1996).

39. Fatma, N., Singh, D.P., Shinohara, T. & Chylack, L.T., Jr. Transcriptional regulation of the antioxidant protein 2 gene, a thiol-specific antioxidant, by lens epithelium-derived growth factor to protect cells from oxidative stress. J. Biol. Chem. 276, 48899–48907 (2001).

40. Yung, E. et al. Specificity of interaction of INI1/hSNF5 with retroviral integrases and

its functional significance. J. Virol. 78, 2222–2231 (2004).41. Priet, S. et al. HIV-1-associated uracil DNA glycosylase activity controls dUTP misin-

corporation in viral DNA and is essential to the HIV-1 life cycle. Mol. Cell 17, 479–490 (2005).

42. Schwartz, O., Marechal, V., Friguet, B., Arenzana-Seisdedos, F. & Heard, J.M. Antiviral activity of the proteasome on incoming human immunodeficiency virus type 1. J. Virol. 72, 3845–3850 (1998).

43. Priet, S., Navarro, J.M., Gros, N., Querat, G. & Sire, J. Functional role of HIV-1 virion-associated uracil DNA glycosylase 2 in the correction of G:U mispairs to G:C pairs. J. Biol. Chem. 278, 4566–4571 (2003).

44. Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

45. Bram, R.J., Hung, D.T., Martin, P.K., Schreiber, S.L. & Crabtree, G.R. Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location. Mol. Cell. Biol. 13, 4760–4769 (1993).

46. Hyberts, S.G. & Wagner, G. IBIS–a tool for automated sequential assignment of protein spectra from triple resonance experiments. J. Biomol. NMR 26, 335–344 (2003).

47. Clore, G.M. & Gronenborn, A.M. Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239, 349–363 (1994).

48. Ferentz, A.E. & Wagner, G. NMR spectroscopy: a multifaceted approach to macromo-lecular structure. Q. Rev. Biophys. 33, 29–65 (2000).

49. Szyperski, T., Neri, D., Leiting, B., Otting, G. & Wuthrich, K. Support of 1H NMR assignments in proteins by biosynthetically directed fractional 13C-labeling. J. Biomol. NMR 2, 323–334 (1992).

50. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

51. Brünger, A.T. X-PLOR Version 3.851: A System for X-ray Crystallography and NMR (Yale University Press, New Haven, Connecticut, 1996).

52. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

53. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55, 29–32 (1996).

A R T I C L E S©

2005

Nat

ure

Pub

lishi

ng G

roup

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tp://

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Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75

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