Interactions between IN and LEDGF/p75 can be monitored at sufficiently high concentrations of these proteins for their biochemical and biophysical analysis. In contrast, SSCs have previously been productively assembled only at very dilute concentrations of donor DNA and IN, which allowed biochemical characterization of concerted integration intermediates (17
), but were not sufficient for biophysical studies. Therefore, our initial experiments focused on scaling-up purification of SSCs. We estimated that ~100-fold scale-up of the previously described preparations was necessary to enable FRET experiments.
Our experimental strategy for preparation of SSCs is depicted in A. Reported reaction conditions for assembly of SSCs (17
) served as a starting point. Recombinant IN and a long viral donor DNA in combination with IN strand transfer inhibitor 118-D-24 (49
) allowed us to effectively trap the SSC. The inhibitor effectively impairs binding of the target DNA to the SSC and prevents formation of the strand transfer complex (18
). To increase the yield of SSCs, reaction volumes were increased 20-fold from 25
µl used in previous studies (17
) to 500
µl in our assays. Using the same buffer conditions as previously reported (17
) IN concentrations in the reaction mixture were optimized as shown in B.
Figure 1. Scaled-up preparations of the SSC. (A) Experimental design. (B) Optimization of IN concentrations for the SSC assembly. Purified SSCs were subjected to SDS–PAGE and the IN band was visualized by western blot. Lane 1, IN load; lane 2, protein markers: (more ...)
Under these conditions, IN forms both specific and non-specific complexes with donor DNAs. To delineate between these, we exploited the intrinsic property of SSCs, which unlike non-specific IN–DNA interactions, are resistant to high ionic strength conditions. The mixture was subjected to treatments with 1
M NaCl followed by size exclusion spin column chromatography. SSCs and free DNAs were readily eluted from the column due to their large molecular weights, while free IN, which dissociated from non-specific DNA sites under high ionic strength conditions, was retained by the column. The obtained fractions were analyzed by SDS–PAGE to monitor relative quantities of IN in the complexes and by non-denaturing agarose gel electrophoresis to determine the purity of the final products.
B shows that the optimal concentration range of IN for the assembly of SSCs under these conditions is 200–400
nM. At these concentrations, free IN is predominantly a dimer (59
) (see also Supplementary Figure S1
and Supplementary Table S1
). At higher protein concentrations, IN forms tetramers (~2
µM) with subsequent concentration increments leading to formation of higher order oligomers and protein precipitation. To delineate the role of IN tetramers in SSC formation, we compared the data in B with IN 3′-processing activities (Supplementary Figure S2
) as pre-assembled IN tetramers are active in this reaction (41
). These experiments revealed a sharp contrast between the 3′-processing activities and the formation of SSCs. The highest 3′-processing activities were detected with 800–1600
nM IN, whereas these protein concentrations were very ineffective for the SSC assembly. These results are consistent with our earlier observations that the highly dynamic interplay between individual IN subunits is essential for productive concerted integration, and that a preformed IN tetramer lacks sufficient flexibility to form the fully functional nucleoprotein complex (41
C compares IN interactions with cognate donor and non-specific DNAs. In line with the previous report (17
) IN formed SSCs resistant to 1
M NaCl treatment only with cognate DNA substrate and not with non-specific DNA (compare lanes 6 and 7 with lanes 4 and 5 in C).
Non-denaturing agarose gel electrophoresis results (D) demonstrate successful scale-up of SSCs (compare lanes 3–5). In the 100-fold scale-up (lane 5), the band corresponding to the SSC was readily detectable with ethidium bromide staining with only residual amounts of dimerized SSCs being observed. These optimized preparations of SSCs were employed for further biophysical analysis.
Previous reports (42
) indicated the importance of the order of viral DNA and LEDGF/p75 addition to IN for effective concerted integration. Particularly puzzling has been the observation that the preformed IN–LEDGF/p75 complex is selectively defective for concerted integration (41–43
). Moreover, the mechanism behind these observations has remained obscure. LEDGF/p75 exhibits dual activities, with its N-terminal domain tightly binding DNA and its C-terminal IBD directly interacting with HIV-1 IN. Each of these properties of the full-length protein could potentially affect SSC formation by different mechanisms. For example, we previously demonstrated that increasing concentrations of LEDGF/p75 effectively competed with HIV-1 IN for viral DNA binding and inhibited the 3′-processing reaction (41
). In contrast, LEDGF/IBD strongly modulated dynamic interplay between individual IN subunits and stimulated the 3′-processing reaction but potently impaired concerted integration (41
To differentiate between these two activities of LEDGF/p75 and to examine how its direct interaction with IN could affect the formation of the SSC, we employed both, the full-length protein and LEDGF/IBD in our studies. Addition of LEDGF/p75 to free IN with subsequent exposure of protein–protein complexes to donor DNA effectively impaired formation of SSCs (A, lane 5). Agarose gel electrophoresis results (B) corroborated with the western blot data. No SSCs were observed when viral DNA was exposed to preformed IN complexes with LEDGF/p75 (B, lane 4). Very similar results were obtained when the above experiments were conducted with LEDGF/IBD instead of the full-length protein (data not shown). Therefore, we conclude that direct interactions of LEDGF/p75 with HIV-1 IN modulate the structure of the retroviral enzyme in a way that impairs formation of the SSC.
Figure 2. Effects of the order of viral DNA and LEDGF/p75 additions to HIV-1 IN on the SSC assembly. (A) SDS–PAGE analysis of SSCs. Lane 1: 1/10 of IN load, lane 2: protein markers: MagicMark XP Western Protein Standard (Invitrogen, Carlsbad, CA, USA), (more ...)
We next examined whether under our reaction conditions, LEDGF/p75 associated with the SSC (C and D). The SSCs prepared according to A were incubated with LEDGF/p75 in a binding buffer containing 750
mM NaCl to prevent non-specific association of the full-length cellular protein with viral DNA. While LEDGF/p75 potently binds DNA in low ionic strength buffers, these interactions are inhibited at NaCl concentrations >200
). Indeed, no binding of LEDGF/p75 with viral DNA was detected in our experiments (D, lane 6). In contrast, 750
mM NaCl did not significantly interfere with LEDGF/p75 binding to IN (see Supplementary Figure S1
) and the cellular protein effectively interacted with the SSC (C, lane 4). Collectively, these results indicate that under our reaction conditions, LEDGF/p75 associated with the SSC through its biologically relevant interactions with IN.
To gain structural insight into how LEDGF/p75 affects IN conformations, we employed protein–protein FRET. The experimental strategy for FRET studies is outlined in . Two separate preparations of IN were used: one labeled with the D and the other with the A fluorophores. The IN concentration range of 200–400
nM was employed, which is optimal for assembly of the SSC (B). At these concentrations, unliganded IN is predominantly a dimer (59
) (see also Supplementary Figure S1
). Upon binding to viral DNA ends, two separate dimers of IN assemble into a tetramer (17
). LEDGF/p75 also promotes IN tetramerization (41
) (see also Supplementary Figure S1
). Assembly of two dimers labeled with D and A fluorophores into tetramers in the presence of viral DNA or LEDGF/p75 is expected to yield a FRET signal. The goal of our experiments was to compare average FRET values for IN–viral DNA and IN–LEDGF/p75 complexes and thereby determine whether IN tetramers formed in these complexes differed from one another.
Figure 3. Scheme illustrating design of protein–protein FRET experiments. Two IN proteins are prepared in parallel: one labeled with the D probe and another with the A probe. The protein concentration range in the reaction mixture is 200–400nM, (more ...)
A crucial step for effective FRET experiments is to site-selectively tether D and A dyes to IN preparations. The Alexa fluorophores chosen for these studies contain reactive maleimide groups enabling covalent attachment to surface Cys residues. Wild-type HIV-1 IN contains 6 Cys residues that present a challenge for site-specific labeling. Of these, C40 and C43 coordinate the structural Zn2+
ion and C130 is partially buried in the structure and not surface accessible. Therefore, these residues were expected to be chemically inert. In contrast, C56, C65 and C280 are surface exposed and could readily react with maleimide groups. Published site-directed mutagenesis studies (63
) showed that each of the three surface cysteines could individually be mutated to Ser without significantly compromising IN catalytic activities in vitro
or viral replication in infected cells. In addition, we also noted that C56 and C65 are proximal to the viral DNA binding channel (33
), and the placement of fluorophores at these locations could potentially interfere with protein–DNA interactions. In contrast, C280 is significantly removed from both viral DNA and LEDGF/p75 binding sites. Therefore, to accomplish the selective tethering of fluorophores, we mutated C56 and C65 to Ser and exploited the reactivity of the native C280 residue sulfhydryl. The data presented in A demonstrate that C280 was indeed specifically targeted by the dye. The C56/C65S variant was readily labeled by both dyes, while no reactivity was observed in the case of the triple C56/65/280
S variant. Importantly, the fluorophore labeled mutant protein used in our FRET studies readily interacted with LEDGF/p75, formed stable synaptic complexes (B) and displayed concerted integration activity (C).
Figure 4. Site-selective labeling of HIV-1 IN with a fluorophore. (A) In parallel experiments, C56/65S and C56/65/280S mutants were subjected to treatment with Alexa 488 maleimide. The reactions were quenched with DTT and subjected to SDS–PAGE. Images of (more ...)
Prior to proceeding with FRET measurements, we monitored time-resolved anisotropies of the D labeled IN variants, both free and complexed with viral DNA or LEDGF/p75 using time-correlated single photon counting. Time-resolved anisotropy curves were well-fit by a single exponential function. Rotational correlation times of 2.2, 2.7 and 2.6
ns were measured for IN alone, IN complexed with viral DNA and IN bound to LEDGF/p75, respectively. These fast rotational correlation times suggest that the probe retained a significant degree of free motion upon its tethering to C280. Importantly, very similar values were observed for IN–LEDGF/p75 and IN–viral DNA complexes (Supplementary Figure S3
), indicating that neither LEDGF/p75 nor viral DNA significantly altered the conformational freedom of the probe on IN. These control experiments assured us that FRET values obtained for unliganded IN and its complexes with LEDGF/p75 and viral DNA can reliably be compared with one another.
To measure FRET between individual IN subunits in the context of various complexes, we initially conducted steady-state (ss) measurements, which reveal average FRET intensities. In the absence of a binding partner, the IN dimers mixed together did not exhibit any detectable FRET signal (A). This was anticipated as at 200
nM concentrations of IN(C280-A) and IN(C280-D) in the reaction mixture, the protein was predominantly a dimer (Supplementary Figure S1
). Furthermore, under such conditions we did not expect to observe significant subunit exchange between two IN preparations for the following reasons. The dissociation constant for IN dimers has been reported to be in the subnanomolar range (59
). Therefore, effective exchange of individual subunits between dimeric forms of IN can only be observed at subnanomolar to low
nM protein concentrations (60
). In contrast, IN forms stable dimers at the protein concentrations of 200–400
nM used here (Supplementary Figure S1
). At the same time, this concentration range is low enough to avoid tetramer formation. IN tetramers and effective exchange of the stable dimers between tetrameric forms of IN can be detected at ~2
µM protein (41
). Therefore, under our assay conditions, the background FRET due to subunit exchange was very minimal (A).
Figure 5. Ss-FRET plots for IN–viral DNA and IN–LEDGF/p75 complexes. (A) IN alone, (B) IN–LEDGF/p75 complex, (C) IN–viral DNA complex, (D) overlay of the spectra from experiments shown in panels A–C. For control experiments, (more ...)
Addition of LEDGF/p75 promoted formation of a tetramer by bridging two IN dimers (41
) (see also Supplementary Figure S1
) and resulted in significant energy transfer (B). In parallel reactions, viral DNA was added to the IN(C280-A) and IN(C280-D) mixture to form the SSC (C). While IN in the context of the SSC is also a tetramer, the IN–viral DNA complex displayed a significantly higher FRET than IN–LEDGF/p75 (see overlay of the spectra in D). These results suggest that tetrameric forms of IN in IN–LEDGF/p75 and IN–viral DNA complexes differ.
We also examined the samples using tr-FRET ( and Supplementary Figure S4
). We noted that donor only controls, where IN(C280-D) was mixed with the unlabeled protein (A) and then incubated with LEDGF/p75 (B) or viral DNA (D), yielded complex decay curves (Supplementary Figure S5
) suggesting that the fluorophore tethered to IN adopts multiple conformations. Potential asymmetric arrangements of individual subunits within multimeric IN could contribute to this. Alternatively, local environment at C280 could allow the fluorophore to adopt multiple conformations. To delineate between these possibilities, we used the isolated CTD as a reliable control. Consistent with previous reports (29
) isolated CTD formed dimers in our experiments as judged by size exclusion chromatography (data not shown). Each symmetrical subunit of this protein fragment contains a single Cys residue at the position corresponding to C280 in the full-length protein (29
). Similarly to full-length IN, the isolated CTD also yielded three exponential decay curves. These findings suggest that the local environment at the tethering site contributes to multiple conformations of the fluorophore. Our results are reminiscent of the published (66
) tr-FRET analysis of Trp residues in proteins indicating that surface tryptophans typically adopt different conformations and yield multi-exponential decays curves. In common with the donor alone, experiments analysis of D–A pairs ( and Supplementary Figure S4
) yielded the decay curves that were best fit to a three exponential decay (Supplementery Figure S5
). No FRET was detected when IN(C280-A) was mixed with IN(C280-D) (A). In contrast, these proteins yielded FRET when incubated with LEDGF/p75 or viral DNA (B and D). Average distances calculated from tr-FRET results were ~81
Å for the IN–LEDGF/p75 complex and ~69
Å for the SSC () indicating distinct IN conformations in these complexes.
Figure 6. Tr-fluorescence decay plots for IN–viral DNA and IN–LEDGF/p75 complexes. (A) IN alone; (B) IN–LEDGF/p75 complex, (C) IN–LEDGF/p75 complex was preformed and then exposed to viral DNA; (D) IN–viral DNA complex; ( (more ...)
We then extended the tr-FRET experiments to test more complex interactions involving IN, viral DNA and LEDGF/p75. The following two pathways for the assembly of large nucleoprotein complexes were considered. First, the IN–LEDGF/p75 complex was preformed and viral DNA was then added. The fluorescence decay profile for this complex was virtually identical to that for the IN–LEDGF/p75 complex ( and ). In the second set of experiments, we first obtained the SSC and then exposed it to LEDGF/p75. The tr-FRET data for this large nucleoprotein complex was very similar to that of the SSC ( and ). The above FRET experiments were also conducted with LEDGF/IBD and the data (see Supplementary Figure S4
) closely mirrored those obtained with full-length LEDGF/p75 (). Taken together, these data show that the conformation of IN tetramer depends on the order of ligand addition.
Our FRET results together with available crystallographic data were employed to generate molecular models for HIV-1 IN interactions with viral DNA and LEDGF/IBD ( and Supplementary Figure S6
). A and B depict two separate conformations of IN tetramers. The model in A was generated stepwise by first modeling HIV-1 IN interactions with viral DNA and then docking LEDGF/IBD into the nucleoprotein complex. Interactions between two functional HIV-1 IN subunits with viral DNA (A) were modeled based on the crystal structure of PFV IN in the complex with cognate DNA (32
) and the resulting nucleoprotein interactions were similar to those proposed recently (33
). However, published studies (32
) did not define NTDs and CTDs in the supporting two subunits. Our FRET data provided complementary information and enabled us to model interactions of the full-length IN tetramer with viral DNA ends (A and Supplementary Figure S6
). As shown in Supplementary Figure S6
(left panels), there are four possible D–A distances. Of these, distances between D2–A2 and D1–A1 pairs are identical due to the 2-fold symmetry between two IN dimers, while the distance between D2–A1 pairs exceeds an effective FRET range (>2
) and would not affect our FRET measurements. Therefore, average FRET distances are likely to be derived from the following three distances D1–A2, D2–A2 and D1–A1. In fact, FRET measurements and molecular modeling results suggest that these three distances are very similar. For example, the D1–A2 distance of ~68
Å is in excellent agreement with the structure-based modeling using the PFV intasome as a template. This distance together with the average FRET distance measurement of ~69
Å for this complex () and the limited length of the loop connecting the CCD and CTD, provided us with significant constraints to position CTDs in supporting subunits as shown in A and Supplementary Figure S6
It should be noted that PFV IN does not interact with LEDGF/IBD and the published model of HIV-1 intasome (33
) did not address its interactions with the key cellular cofactor. Our data show that LEDGF/p75 potently binds the SSC through its biologically relevant site on HIV-1 IN (D) and that the cellular protein does not alter the pre-formed architecture of the nucleoprotein complex ( and ). In complete agreement with these experimental data, we were able to dock two LEDGF/IBD molecules onto the SSC without altering the pre-existing IN–viral DNA interactions (A). In particular, each LEDGF/IBD engages the CCDs of one dimer and establishes additional contacts with the NTD from another dimer, which ensures high-affinity binding of the cellular cofactor to the complex (41
). Taken together, the experimental results presented here have allowed us to extend previous modeling of HIV-1 IN–viral DNA interactions (33
) by building a ternary complex between viral DNA, full-length IN tetramer and LEDGF/IBD (A).
B shows an alternative conformation of the IN tetramer in complex with LEDGF/IBD. To create this model, we considered the following main criteria. Our published biochemical studies (41
) have shown that interactions between the NTD and the CCD are important for IN tetramerization and high-affinity LEDGF/p75 interactions. In agreement with these findings, subsequent crystallographic studies (52
) with the two domain construct of MVV IN have demonstrated that LEDGF/IBD bridges between the NTD of one dimer and the CTD of another dimer. Therefore, these interactions were included in our model. Published studies (41
), however, did not address the positioning of the CTDs in the protein–protein complex. Therefore, our FRET data () were employed to create the model between full-length IN and LEDGF/IBD ( and Supplementary Figure S6
). Additional constraints were provided by SAXS data (56
), which revealed the molecular shape and global dimensions for the full-length HIV-1 IN complex with LEDGF/IBD. The resulting model and calculated distances for D–A pairs are given in A and Supplementary Figure S6