Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein

Abstract

The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.

FIGURE 1.

The LEDGF proteins used in our studies. The upper wild-type protein exhibits two distinct chromatin and IN binding activities (green and blue, respectively). The middle D366N mutant (mt) protein is severally impaired for IN binding (47, 50). In contrast, the IBD interacts with IN but lacks the ability to bind DNA and chromatin (38, 40).

FIGURE 2.

Effects of LEDGF and the isolated IBD on IN 3′-processing and DNA strand transfer activities. A, schematic of oligonucleotide-based integration assay. B, experimental results. The positions of 21-mer substrate (21-mer S) and the products of 3′-processing (19-mer P) and strand transfer (STP) reactions are indicated. Lanes 1, 7, and 13, 21-mer DNA substrate; lanes 2, 8, and 14, 50 nm DNA + 0.5 μm IN. The following concentrations of the indicated LEDGF proteins were included in the reactions: lanes 3, 9, and 15, 0.25 μm, lanes 4, 10, and 16: 0.5 μm, lanes 5, 11, and 17:1 μm, and lanes 6, 12, and 18:2 μm. C, schematic diagram of concerted integration assay. Integration of one donor (D) DNA into the target plasmid yields half-site (HS) or single-end integration products. Integration of a pair of D ends into both strands of target DNA results in full-site (FS) or concerted integration. The D can otherwise integrate into separate D molecules to form D-D products. D, agarose gel image of reaction products. Lane 1, ³²P-labeled lambda/HindIII DNA mass markers; lane 2, control reaction without IN; lane 3, reaction with 400 nm IN; lanes 4, 5, and 6, contained 0.25, 0.5, and 1.0 μm IBD, respectively; lanes 7, 8, and 9 contained 0.25, 0.5, and 1.0 μm LEDGF, respectively.

FIGURE 3.

Subunit exchange assay using His-tag IN and SDS-PAGE: design (A) and results (B). A, subunit exchange between IN multimers was tested by mixing two different IN proteins: IN1, a tag-free form and IN2, containing the 6× His tag at its C terminus. The full-length proteins are depicted as dimers (IN1-IN1) and (IN2-IN2). Both proteins contained wild-type IN sequences and displayed identical catalytic activities. After mixing, unbound proteins were washed from the resin and bound complexes were analyzed by SDS-PAGE. IN1 and IN2 were clearly separated based on their molecular weight differences. B, data in lanes 1–4 indicate that IN2 was able to quantitatively pull-down tag-free IN1. The recovered multimers contained a mixture of IN2-IN2 and IN1-IN2, while tag-free IN1-IN1 was washed out from the Ni-NTA resin. To assess the impact of LEDGF on IN subunit exchange, the IN2 multimer was preincubated with LEDGF and then exposed to IN1 (lanes 5–8). The results show that LEDGF interacted with IN2 and effectively prevented IN subunit exchange (lanes 5–8). In contrast, mtLEDGF did not bind IN2 or affect subunit exchange (lanes 9–12). Total amounts of input IN1, IN2, LEDGF, and mtLEDGF proteins are shown in lanes 13, 14, 15, and 16, respectively.

FIGURE 4.

Size exclusion chromatography of free IN and IN-IBD complexes. 2.5 μm IN and 5 μm IBD were used in these experiments. A, chromatograms of wild-type IN protein in its free form (top) and complexed with IBD (bottom). B, elution profiles of the soluble double mutant (F185K/C280S) IN protein in its free form (top) and complexed with IBD (bottom). Peaks corresponding to tetramer (Tet) IN, dimer (Dim) IN, as well as IBD-bound IN tetramer (Tet+IBD) and dimer (Dim+IBD), are indicated.

FIGURE 5.

Upper panel, schematic presentation of the protein footprinting strategy. The structures of the CCD and the IBD-CCD complex are used for illustration while the experiments were performed with full-length IN and LEDGF. In parallel experiments free IN and the IN-LEDGF complex were subjected to treatment by small chemical modifiers (M). Surface residues in free IN and the complex were modified, but the interacting amino acids in the complex were shielded from modification. His-tag LEDGF and tag-free IN proteins were used. Following the modification reactions, the complex was pulled-down by NTA beads, which enabled recovery of only the LEDGF-bound form of IN from the reaction mixture. The interacting proteins were then separated by SDS-PAGE. The IN band was excised and subjected to in-gel proteolysis. Subsequent comparative mass spectrometry (MS) analyses revealed modification patterns in free protein and the complex. Lower panel, representative segments of the MALDI-ToF mass spectra. A, free IN was treated with HPG or NHS-biotin. B, IN was preincubated with mtLEDGF and then exposed to treatments with HPG or NHS-biotin. C, IN-LEDGF complexes were preformed and then exposed to HPG or NHS-biotin treatments. Start and end amino acid numbers of the detected peptide peaks are indicated. IN residues affected by modification are depicted in brackets.

FIGURE 6.

Biochemical characterization of IN mutant proteins. A, size exclusion chromatography of wild type and indicated mutants. All protein concentrations were 10 μm. The elution time points corresponding to tetrameric and dimeric IN are indicated by arrows. B, Western blot results for LEDGF binding to wild type and mutant IN proteins. Increasing concentrations of LEDGF were incubated with 100 nm of the indicated IN. The concentrations of LEDGF were as follows: lane 1: 50 nm, lane 2: 100 nm, lane 3: 150 nm, lane 4: 200 nm, lane 5: 250 nm, lane 6: 300 nm, lane 7: 350 nm, lane 8: 400 nm, lane 9: 500 nm, lane 10: 650 nm. C, quantitative analysis of the Western blot results. D, 3′-processing and DNA strand transfer activities of the indicated IN proteins; other labeling is the same as in Fig. 2B.

FIGURE 7.

A model for the IBD bound NTD-CCD tetramer. Two available crystal structures of the NTD-CCD tetramer and the IBD-CCD complex were superimposed. Individual subunits of IN are colored cyan, green, yellow, and orange. The acidic residues, which coordinate catalytic metals, in the green and yellow subunits are in spheres and colored red. For clarity only Lys-14 in the green subunit and the basic triad (Lys-186, Arg-187, Lys-188) in the yellow subunit are depicted as spheres. These residues contribute to interactions between the two cyan-green and yellow-orange dimers. The two IBD molecules colored magenta establish symmetrical high affinity interactions, bridging the two dimers together. The lower part of the picture shows that the IBD bound to the CCDs of the yellow-orange dimer is positioned to interact with the α-helix within the green NTD that contains Lys-14. The side chains of Asp-6, Glu-10, and Glu-13 in the green NTD are depicted as red sticks. These amino acids potentially establish charge-charge interactions with the side chains of Lys-401, Lys-402, Arg-404, and Arg-405 (blue sticks) of the IBD helix. The additional two lower affinity binding IBD molecules (gray), which coordinate the CCDs of either cyan-green or yellow-orange dimers, are significantly distanced from the tetramer interfaces.