Binding stoichiometry and structural model of the HIV-1 Rev/importin β complex

Abstract

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin β (Impβ), but how Rev associates with Impβ is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impβ/Rev complex. We show that Impβ binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impβ cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impβ-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impβ cargos.

Figure 1.. Rev structure and dimerization interfaces.

(A) Scheme depicting the nuclear import cycle of Rev by Impβ. (B) Domain organization of Rev. (C) Oligomerization interfaces reported for Rev. Residues mutated to destabilize the A–A and B–B interfaces are indicated in cyan.

Figure 2.. HIV-1 Rev forms a stable complex with Impβ.

(A) Analysis of different Rev constructs by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) using a Superdex 75 10/300 GL column. Elution curves recorded at 280 nm and molar mass distributions determined by MALLS are shown for the following Rev constructs: WT (green), V16D (blue), V16D/I55N (purple), and Rev^4-69(V16D/I55N) (pink). The molar masses detected at the elution peaks are indicated. (B) SEC analysis of an Impβ/Rev^OD complex. Top: Elution profiles of samples containing Impβ (blue), Rev^OD (magenta), or a mixture of Impβ and Rev^OD (green). Fractions collected are indicated in brown. Chromatography was performed using a Superdex 200 5/150 Increase GL column. Bottom: SDS–PAGE analysis of the indicated fractions. (C) SEC analysis of an Impβ/Rev^WT complex. Elution profiles are shown for Impβ (blue), Rev^WT (magenta) and a mixture of Impβ and Rev^WT (green). Because most free Rev^WT forms insoluble aggregates in the buffer conditions used and hence is not detected in the elution, the elution profile of Rev^WT analyzed on a column pre-equilibrated in high-salt buffer was included as an additional reference (purple). (D) Native gel analysis showing the association of Impβ with one or more monomers of Rev^WT (top) and Rev^OD (bottom). (E) SEC/MALLS analysis of Impβ in the presence and absence of Rev. Elution curves recorded at 280 nm and molar mass distributions determined by MALLS are shown for His-tagged Impβ in the absence (blue) and presence of a either a twofold (light green) or fourfold (dark green) excess of Rev^WT. The observed masses of 124 and 137 kDa are consistent with those expected for 1:1 (116.2 kDa) and 1:2 (129.4 kDa) Impβ:Rev stoichiometry. Chromatography was performed using a Superdex 200 10/30 GL column. Injected concentrations were 20 μM Impβ and 40 or 80 μM Rev^WT.

Figure S1.. Glutaraldehyde cross-linking and MALDI-TOF mass spectrometry indicate that Impβ binds two Rev monomers.

(A) Impβ was incubated with Rev^OD in the presence of glutaraldehyde and the mixture was analyzed by native (top panel) and denaturing (bottom two panels) gel electrophoresis followed by Coomassie staining. The upper denaturing gel (containing 15% acrylamide:bis-acrylamide in a 37.5:1 ratio) allowed Impβ and Rev to be visualized on the same gel, whereas the lower gel (containing 10% acrylamide:bis-acrylamide) was used to resolve cross-linked species migrating close to Impβ. At Rev^OD concentrations yielding a single gel shift by native gel electrophoresis, SDS–PAGE analysis revealed an additional band that migrated just above Impβ (bottom gel, compare lanes 1 and 3). At higher Rev^OD concentrations that yielded a supershift on the native gel, a second more slowly migrating band was detected by SDS–PAGE (bottom gel, lanes 4–5). The vertical black line on the native gel indicates that lanes 1–2 and lanes 3–5 are taken from nonadjacent regions of the same gel. (A, B) MALDI-TOF mass spectrometry analysis of samples corresponding to lanes 3 (upper profile) and 5 (lower profile) from panel (A) was performed. MALDI MS spectra showed peaks consistent with the molecular mass of free Impβ (indicated by a circle), Impβ cross-linked to either one or two Rev^OD monomers (indicated by a circle attached to one or two diamonds, respectively), or cross-linked Rev^OD dimers (indicated by two diamonds).

Figure S2.. Mass determination of Impβ and Rev proteins under denaturing conditions.

A, B) LC/ESI-TOF MS analysis of (A) Rev^OD and (B) Impβ. The upper mass spectrum in each panel represents the raw ESI data, whereas the lower panel shows the deconvoluted ESI spectrum. βME, β-mercaptoethanol; TFA, trifluoroacetic acid.

Figure 3.. Native MS reveals that Impβ binds up to two Rev monomers.

(A) Summary of masses observed by native MS. (B, C, D, E, F, G) Native MS spectra. Peaks labeled by magenta circles correspond to unbound Impβ. Peaks labeled by blue circles with a single dot or by green circles with two dots correspond to Impβ/Rev complexes with 1:1 or 1:2 stoichiometry, respectively. (B) Spectrum of Impβ in the absence of Rev. (C, D) Spectra of Impβ incubated with a (C) twofold or (D) fivefold molar equivalent of Rev^OD. (E) Spectra of Impβ in the presence of the truncated construct Rev^ODΔ. (F, G) Spectra of Impβ incubated with a (F) twofold or (G) fivefold molar equivalent of Rev^WT.

Figure S3.. RanGTP competes with both monomers of Rev for binding to Impβ.

(A, B) Samples containing a mixture of Impβ, RanGTP and either (A) Rev^OD or (B) Rev^WT were analyzed by native gel electrophoresis (top) or SDS–PAGE (bottom) followed by Coomassie blue staining. Protein concentrations used were 2.5 μM for Impβ, 5 μM for RanGTP and either 2.5 or 5 μM for Rev proteins, indicated by (+) and (++), respectively. Compared with unbound Impβ (lanes 1, 11 and 18), incubating Impβ with Rev resulted in shifted and supershifted bands corresponding to 1:1 and 1:2 Impβ/Rev complexes, marked by light and dark green arrowheads, respectively (lanes 4, 7, 9, 14, and 16). Incubating Impβ with RanGTP yielded a band that migrated with intermediate mobility (purple arrowhead, lane 5). When all three Impβ, RanGTP, and Rev proteins were co-incubated, the bands corresponding to the Impβ/Rev complexes were absent or only fainted detected whereas strong intensity was observed for the Impβ/RanGTP band, showing that RanGTP prevents Impβ from binding either monomer of Rev.

Figure 4.. Rev binds Impβ at two sites with sub- and low micromolar affinity.

(A, B) Representative isothermal titration calorimetry profiles of the binding of Impβ to (A) Rev^OD and (B) Rev^ODΔ. Top: Differential power time course of raw injection heats for a titration of Impβ into the Rev protein solutions. Bottom: Normalized binding isotherms corrected for the heat of dilution of Impβ into buffer. The solid line represents a nonlinear least squares fit using a model consisting of two nonsymmetric classes of binding sites. (C, D) Thermodynamic values obtained from isothermal titration calorimetry data for the binding of Impβ to (C) Rev^OD and (D) Rev^ODΔ. Binding to site 1 is characterized by a favorable enthalpy (ΔH ≈ −8 kcal/mol) and a negligible entropy change for both Rev^OD and Rev^ODΔ, whereas binding to site 2 is associated with an unfavorable entropy change that is offset by a large negative enthalpy change. Thermodynamic parameters (in kcal/mol) are as follows: Rev^OD: ΔG = −8.3 ± 0.1 and −7.2 ± 0.4, ΔH = −6.3 ± 2.4 and −26.5 ± 3.5, −TΔS = −2.1 ± 2.5 and 19.3 ± 3.9 for sites 1 and 2, respectively; Rev^ODΔ: ΔG = −8.5 ± 0.2 and −7.2 ± 0.8, ΔH = −8.3 ± 0.6 and −23.6 ± 3.6, −TΔS = 0.3 ± 0.8 and 16.5 ± 4.0 for sites 1 and 2, respectively. Data represent the mean ± SD from three independent replicates. All replicate profiles are shown in Fig S4.

Figure S4.. ITC analysis of the Impβ:Rev interaction.

(A, B) ITC profiles of the binding of Impβ to (A) Rev^OD and (B) Rev^ODΔ. Three independent replicates are shown for each assay. Top: Differential power time course of raw injection heats for a titration of Impβ into the Rev protein solutions. Bottom: Normalized binding isotherms corrected for the heat of dilution of Impβ into buffer. The solid line represents a nonlinear least squares fit using a model consisting of two nonsymmetric classes of binding sites. Profiles labeled “Exp1” are identical to the panels shown in Fig 4A and B.

Figure S5.. Isothermal titration calorimetry data are consistent with two classes of Rev binding sites on Impβ.

Representative isothermal titration calorimetry profile for the binding of Impβ to Rev^OD showing that a model consisting of two nonsymmetric classes of binding sites (right) yields a better fit to the normalized binding isotherm than a model consisting of a single class of binding site (left), as highlighted by the data points circled in red.

Figure 5.. NMR analysis of Rev binding by Impβ.

(A) ¹H,¹⁵N HSQC spectrum of free Rev^OD (600 MHz, 283K). (B) ¹H,¹⁵N-HSQC spectrum Impβ-bound Rev^OD (blue) superimposed on that of unbound Rev^OD (green). (C) Secondary chemical shifts (96) of unbound Rev^OD. The two point mutations are indicated by asterisks. (D) Ratio of peak intensities for Rev residues in the presence and absence of Impβ (600 MHz, 283K).

Figure 6.. Impβ retains an extended conformation upon binding Rev.

(A) Top: Representative crystal structures of Impβ illustrating different degrees of compaction of the HEAT-repeat array. Bottom: Model-independent parameters obtained from SAXS data compared with values calculated for available Impβ crystal structures using the programs CRYSOL (127) and PRIMUS (126). All structures are of human Impβ except for 1UKL which is of murine Impβ. Chain A in 3W5K lacks coordinates for Impβ residues 1–15. For the calculation of R_g, D_max, and χ² values, this chain was extended to include these residues (denoted A*) by replacing HEAT repeat 1 (residues 1–31) by the corresponding residues from 1UKL following local alignment of the two structures. (B) Scattering data from unbound Impβ (black) and the Impβ/Rev^ODΔ complex (red). (C) Scattering data from unbound Impβ (black) compared with profiles calculated from representative cargo-bound conformations exhibiting different degrees of elongation (colored lines).

Figure 7.. Cross-linking-MS localizes two Rev binding regions on Impβ.

(A) Graphical summary of cross-links. Impβ-Rev cross-links involving the N-terminus or Lys20 residue of Rev are shown in green and red, respectively. Rev–Rev and Impβ-Impβ cross-links and monolinks are shown in blue. Red, green, and blue inverted flags indicate Impβ residues that form cross-links with Rev Lys20 (group 1), with both Rev Lys20 and the Rev N-terminus (group 2), or for which no cross-links with Rev were detected (group 3), respectively. The 19 HEAT repeats (HR) of Impβ are indicated. (B) Impβ Lys residues modified by BS3 and detected in cross-links or monolinks. Group-1, -2, and -3 lysines are highlighted in red, green, and blue, respectively. For group-1 and -2 lysines, the number of peptide spectrum matches (PSMs) and the best pLink E-value score are indicated. (C) Surface representation of Impβ showing the location of cross-linked group-1, -2, and -3 Lys residues, colored red, green, and blue, respectively. Shading is from dark to light gray from N- to C-terminus. (D) Solvent-accessible surface distances (SASDs) between pairs of Impβ group-1 Lys residues. The upper and lower triangles show distances for the conformations of Impβ bound to the importin α IBB domain (PDB 1QGK) and to SREBP-2 (PDB 1UKL), respectively. SASDs over 70 Å are outlined in dark blue. Distances were calculated using the Jwalk webserver (101). The SAS distances shown here and in Fig S6B suggest that Lys23, Lys62, and Lys68 cross-link with Rev at the N-site and that Lys854, Lys857, Lys859, Lys867, and Lys873 cross-link with Rev at the C-site. (E) Spheres of radius 35 Å centered on the Cα atoms of group-1 residues Lys23 and Lys873 show that BS3 molecules bound to these two lysines cannot cross-link to the same Rev Lys20 position. (F) Localization of the Cα atom of Rev Lys20. Green and magenta volumes show the N- and C-terminal regions of space within cross-linking distance of group-1 residues in either HEAT repeats 1 and 2 (K23, K62, K68) or repeat 19 (K854, K857, K859, K867, and K873), respectively, defined by the intersection of 35 Å spheres centered on the Cα atoms of these residues. If the centroid of each envelope is used to estimate the position of the Rev K20 Cα atom, then the positional uncertainty, calculated as the rmsd of each grid point within the envelope (sampled on a 2 Å grid) relative to the centroid, is 22.8 Å for the N-site and 21.6 Å for the C-site for the 1UKL conformation. (C, E, F) The Impβ conformation shown in panels (C, E, F) is that of SREBP-2-bound Impβ (PDB 1UKL).

Figure S6.. Cross-linking-MS distance constraints localize two Rev binding regions on Impβ.

(A) Spheres of radius 35 Å centered on the Cα atoms of group-1 residues Lys23 and Lys873 show that BS3 molecules bound to these two lysines cannot cross-link to the same Rev Lys position regardless of the Impβ conformation. The four conformations shown are those of Impβ bound to the following cargos: the IBB domain of Snurportin1 (PDB 3LWW chain C), the IBB domain of importin α (PDB 1QGK), the SREBP-2 complex (PDB 1UKL) and Snail1 (PDB 3W5K). (B) Distances between pairs of Impβ group-1 Lys residues. The upper and lower triangles show distances for the conformations of Impβ bound to the IBB domain of importin α (PDB 1QGK) and the SREBP-2 complex (PDB 1UKL), respectively. Solvent-accessible surface distances (SASDs) over 70 Å are outlined in dark blue. Distances were calculated using the Jwalk webserver (101). (C) Localization of the Cα atom of Rev Lys20. Green and magenta volumes show the N- and C-terminal regions of space within cross-linking distance of group-1 residues in either HEAT repeats 1 and 2 (K23, K62, K68) or repeat 19 (K854, K857, K859, K867, and K873), respectively, defined by the intersection of 35 Å spheres centered on the Cα atoms of these residues.

Figure 8.. Impβ recognizes Rev peptides derived from helix α2 and the α1-α2 loop.

(A) Examples of thermal denaturation curves measured by differential scanning fluorimetry of Impβ in the presence and absence of Rev peptides. Data are shown for Rev peptides 9–11. The melting temperature (T_m) and difference in T_m compared with unbound Impβ (ΔT_m) are listed as mean values ± SD from three independent experiments. (B) Summary of ΔT_m values determined by differential scanning fluorimetry analysis of Impβ for all 27 peptides spanning the Rev sequence. Details are shown for peptides 7–12. (C) Structure of the Rev N-terminal domain highlighting the residues in helix α2 and the α1-α2 loop spanned by Rev peptides 9–11. Atomic coordinates are taken from PDB 2X7L (69).

Figure 9.. Charge-reversal mutations identify an Impβ-binding epitope on Rev.

(A) Rev double and triple R/K→D substitution mutations. (B) Competitive fluorescence polarization (FP) inhibition assays showing the ability of WT and mutant forms of Rev to displace a fluorescently labeled Rev-NLS peptide from Impβ. A representative experiment is shown for each protein. Data shown are mean and SD values from two technical replicates. (C) Plot of IC₅₀ values derived from FP inhibition assays for WT Rev and double and triple mutants. ****P ≤ 0.001; **P ≤ 0.01. P-values were determined using an ordinary ANOVA test. (D) Plot of IC₅₀ values for WT Rev and single R→D point mutants. (E) Summary of IC₅₀ (mean ± SD) and corresponding pIC₅₀ values. N represents the number of biological replicates. ΔpIC₅₀ values are reported relative to WT Rev. (F) View of the Rev helical hairpin showing the Arg residues selected for single-point mutations. Carbon atoms are colored from blue to red in order of increasing ability of the R→D substitution to compromise Impβ recognition, as measured by ΔpIC₅₀ values.

Figure 10.. Compensatory effects between charge-reversal mutants of Impβ and Rev.

(A) Multiple point mutants of Impβ in which 2–4 Asp or Glu residues are replaced by Arg residues. (B) IC₅₀ values obtained from FP inhibition assays measuring the ability of WT Rev to displace a Rev-NLS peptide from WT or mutant forms of Impβ. Results from individual assays are shown together with the mean. (C) The same assay performed with WT and charge-reversal mutant forms of Rev. Data for Rev mutants are colored as in Fig 8. Data shown are mean and SD values from 4 to 14 biological replicates (see also Table S2). (D) Compensatory effects between charge-reversal mutants. An interaction between an acidic residue on Impβ and an Arg residue on Rev is disrupted by a charge-reversal (R→D) mutation of Rev. The interaction is restored by a charge-reversal (D/E→R) mutation on Impβ. (E) Derivation of ΔΔpIC₅₀ values. Top: FP inhibition assays performed with WT Rev (black curves) or the R4 mutant (red curves) together with WT Impβ (circles) or the B2 mutant (diamonds). Bottom: The ability of WT or mutant Rev to displace the Rev-NLS peptide from WT or mutant Impβ is plotted as pIC₅₀ values and as the shift (ΔpIC₅₀) relative to the value observed when WT Rev is assayed with WT Impβ. The ΔΔpIC₅₀ value represents the shift in ΔpIC₅₀ observed when the assay is performed with an Impβ mutant instead of WT Impβ. A positive value of ΔΔpIC₅₀ indicates that the tested Rev mutant more potently displaces the Rev-NLS peptide from the indicated Impβ mutant than from WT Impβ. (F, G) Summary of ΔΔpIC₅₀ values for the indicated combinations of Impβ and Rev proteins. The dotted line at ΔΔpIC₅₀ = 0.1 (corresponding to a 21% decrease in IC₅₀ between the mutant and WT Impβ) indicates the threshold used to identify compensatory mutations. Compensatory effects satisfying this criterion involving single-point mutants of Impβ and Rev are marked by a gray arrow and numbered 1–8. For all eight of these mutant combinations, the mean shift in IC₅₀ value observed with the Impβ single-point mutant relative to WT Impβ was statistically significant according to a Dunnett’s multiple comparisons test (P-values for mutant combinations 1–8 were <0.0001, 0.0002, 0.0001, 0.0022, 0.0368, 0.0084, <0.0001 and 0.0127, respectively). (H) Summary of compensatory interactions involving single Impβ and Rev residues. (G) The numbered arrows correspond to the mutant combinations numbered 1–8 in (G). The thickness of arrows is proportional to the ΔΔpIC₅₀ values of the interacting mutants.

Figure S7.. Compensatory effects between charge-reversal mutants of Impβ and Rev.

Panels (A, B, C, D, E, F, G) show representative individual FP inhibition assays performed with WT Rev (black curves and symbols) or the indicated Rev mutant (colored curves and symbols) together with WT Impβ (circles) or the indicated Impβ mutant (squares, triangles or diamonds).

Figure S8.. Electrostatic interactions suggested by compensatory mutagenesis data are more likely to involve Rev bound at the C-site than at the N-site.

Panels (A, B, C, D) show the results of HADDOCK docking experiments 1–4, respectively, described in the text. In each case, the lowest-energy structure (LES) for the top-ranking cluster of docking solutions is shown, with Impβ colored from blue to cyan from N- to C-terminus and Rev in violet. In panel (B), the LES from the second ranked cluster is also illustrated, with Rev shown in gray. The pink and blue arrows indicate the orientation (from N- to C-terminus) of Rev helix α2 and the Impβ superhelical axis, respectively. BS3 cross-linking and electrostatic interaction restraints included in each experiment are summarized (see Table S3A for details). (E) Evidence supporting the conclusion that electrostatic interactions deduced from our compensatory mutagenesis data are more compatible with the C-site of Impβ than with the N-site. Experiments 3 and 4 are identical to experiments 1 and 2, respectively, except that the Glu437^Impβ:Arg48^Rev interaction was included as an additional distance restraint. (B, D) Experiments 2 and 4 yielded similar results (Table S3B), except that experiment 4 resulted in a greater number of clustered solutions (189 versus 177) that were more uniform in configuration (compare Fig S9D with Fig S9B) and yielded a top-ranked solution in which residues Glu437^Impβ and Arg48^Rev were closer together (compare panel (D) versus (B)). In contrast, experiment 3 yielded a different outcome compared with experiment 1 (Table S3B), resulting in fewer clustered solutions (84 versus 143) that were more diverse in configuration (compare Fig S9C with Fig S9A). (A, C, D) Indeed, several clusters of solutions (including the top-ranked) positioned Rev next to HEAT repeats 7–19 with helix α2 parallel to the Impβ superhelix, that is, closely resembling the C-site solution for experiment 4 (panels [A, C, D]), even though the BS3 cross-linking restraints used were those associated with the N-site. Moreover, all the solutions that placed Rev in the N-site (clusters ranked second and fourth) positioned residues Glu437^Impβ and Arg48^Rev far apart. These findings suggest that the set of electrostatic interactions used as distance restraints in experiments 3 and 4 are more compatible with the C-site than with the N-site.

Figure S9.. Results of docking experiments with program HADDOCK.

Panels (A, B, C, D) show the results for experiments 1–4, respectively. For each docking experiment, the 200 lowest-energy binding configurations were grouped into clusters according to structural similarity. Clusters were then ranked according to the average energy of the four lowest-energy structures in each cluster. The four lowest energy structures are shown for each cluster. All members of each cluster are colored according to the color scheme shown at the left. The downward gray arrow indicates the direction of the Impβ superhelical axis. For each cluster, the approximate binding orientation of Rev is designated by indicating the orientation of Rev helix α2 as roughly antiparallel (↑), parallel (↓) or perpendicular (← or →) relative to the Impβ superhelical axis. Rev monomers with parallel or antiparallel orientations are shown as ribbon diagrams, and those with perpendicular orientations are shown as Cα line tracings. Although the inclusion of the additional distance restraint between Glu437^Impβ and Arg48^Rev in experiment 3 results in a greater diversity of cluster configurations relative to experiment 1, its inclusion in experiment 4 results in more uniform clusters relative to experiment 2.

Figure S10.. Analysis of distances between Impβ and Rev residues in rigid-body docking simulations.

(A) Boxplots of Cβ-Cβ distances between the indicated Impβ and Rev residues for docking simulations involving the N-site (green) or C-site (purple). (B) Scatter plot of docking configurations showing the distribution of Cβ-Cβ distances between residues D288^Impβ and Arg42^Rev (horizontal ordinate) and between residues E437^Impβ:Arg48^Rev (vertical ordinate) for docking simulations involving the N-site (left panel) or C-site (right panel). The number of configurations satisfying the indicated distance cutoffs is indicated in blue. (C) Scatter plot of docking configurations showing the distribution of Cβ-Cβ distances between residues Lys206^Impβ and Lys20^Rev (horizontal ordinate) and between residues Lys537^Impβ:Lys20^Rev (vertical ordinate) for docking simulations involving the N-site (left panel) or C-site (right panel). The number of configurations satisfying the distance cutoffs (indicated by dashed blue lines) is shown in blue. Because the Lys20^Rev Cβ atom was centered on each grid position before applying the rotational search, each point in the plot may correspond to several different (up to 4,056) Rev orientations.

Figure 11.. Structural model of Rev bound to Impβ at the C-site.

(A) BS3 cross-links and electrostatic interactions used as distance constraints for rigid body docking and as interaction restraints in program HADDOCK. (B) Configurations of the Impβ/Rev complex obtained by rigid body docking. The mean variation in Rev orientation and centroid position are 33° and <5 Å, respectively, relative to the top-ranked configuration (see also Table S4). (C) Configurations of the Impβ/Rev complex obtained using HADDOCK. For clarity, only 28 (the four lowest-energy structures in each of the seven clusters) of the 194 clustered solutions are shown. Solutions are colored according to the rank of the corresponding cluster. The mean variation in Rev orientation and centroid position are 34° and 4 Å, respectively (see also Table S5). (D) Superposition showing agreement between docking solutions obtained by rigid body sampling and by HADDOCK. (E) “Average” configuration of the Impβ/Rev complex that minimizes the deviation from models obtained by rigid-body and HADDOCK docking experiments. Compared with the average configuration, the Rev orientation in the individual rigid-body and HADDOCK configurations shows the greatest variation (by a maximum of 52°) in the angle about the long axis of Rev (roll angle) and smaller variations (up to 25–35°) about the two orthogonal angles (yaw and pitch). The approximate location of the Rev CTD is indicated. Upper inset: Principal axes and corresponding rotational angles of Rev. Lower inset: Centroids of Rev monomers obtained in rigid-body (green spheres) and HADDOCK (violet spheres) docking experiments. These centroids differ from that of the average model by a maximum of 5 or 6 Å along each of the principal axes of Rev.

Figure S11.. Top-ranking configurations from docking experiments.

(A) Plot of solvent-accessible surface (SAS) distances calculated for the top-ranking configuration obtained by rigid-body sampling (top) or HADDOCK (bottom) analysis. SAS distances were calculated between Rev residue Lys20 and Impβ lysine residues belonging to group 1 (red), group 2 (green), or group 3 (blue), as defined in Fig 7B. Group 1 lysines associated with the N- or C-site of Impβ are indicated by open and closed red circles, respectively. SAS distances were calculated using the Jwalk server (101). (B) Orthogonal views of the top-ranking configuration obtained by rigid-body sampling (left) or HADDOCK (right) analysis. Acidic Impβ residues and basic Rev residues hypothesized to be in close proximity on the basis of compensatory charge-reversal mutation data are shown in stick representation. In the bottom panels, Rev helix α1 is omitted for clarity.

Figure S12.. Comparison of docking solutions.

(A) Top: Structures of the top-ranking solution from the rigid-body docking and HADDOCK experiments are shown and the “average” configuration of all solutions obtained by the two docking methods. Bottom: Table summarizing the relative shift in the centroid position of Rev and its relative rotation (polar angle κ) in pairwise comparisons of the three structures, calculated using program Lsqkab from the CCP4 suite (128). (B) Superposition of the three Rev docking poses shown in (A).

Figure S13.. Comparison of structural models of the 1:1 Impβ/Rev complex with SAXS data.

(A) Scattering data from the Impβ/Rev^ODΔ complex compared with the scattering profile calculated from the “average” docking configuration of Rev bound to the Impβ C-site. (B) Replacing the Impβ conformation from PDB 1UKL with the slightly more elongated conformation from PDB 3W5K yields an improved agreement between the observed and calculated scattering profiles. (C) Comparison of the two Impβ conformations of PDB 1UKL with 3W5K. The 3W5K conformation is more extended by ∼3 Å along the HEAT-repeat superhelical axis.

Figure S14.. Configurations for the binding of Rev monomer 2.

(A, B) Unlikely configurations in which Rev monomer 2 (cyan) interacts with Rev monomer 1 (magenta) via an A–A (head-to-head) or B–B (tail-to-tail) interaction. Predicted distances between residue Lys20 on Rev monomer 2 and Impβ residues Lys23, Lys62, and Lys68 are roughly twice the BS3 cross-linking distance constraint. (C) Results of the rigid body docking analysis showing the centroids of Rev monomer 2 that are consistent with BS3 cross-linking constraints associated with the N-site and sterically compatible with Impβ bound to Rev monomer 1 via the C-site. Centroids of Rev molecules within and beyond contact distance of Rev monomer 1 are indicated by blue and green spheres, respectively. (D) Two representative orientations of Rev monomer 2 in which the N-and C-termini of the Rev helical hairpin are near the N-terminal end of Impβ (N-ward) or are located midway between the N- and C-terminal ends (sideward). (E) Hypothetical configuration showing that the binding of Rev to the N- and C-sites of Impβ is compatible with a C–C interaction between the two Rev monomers as observed in PDB 5DHV (67).

Figure S15.. Comparison of structural models of the 1:2 Impβ/Rev complex with SAXS data.

(A, B) Scattering data of the Impβ/Rev^ODΔ complex were compared with scattering profiles calculated for representative docking configurations of the 1:2 Impβ/Rev complex, using Impβ conformations from (A) PDB 1UKL and (B) PDB 3W5K. The placement of Rev monomer 1 is that of the “average” configuration shown in Fig 11E. The placement of Rev monomer 2 corresponds to that of the antiparallel, C–C homodimer and transverse arrangements shown in Fig S14D. For all three arrangements, the 3W5K conformation yielded a better fit compared with 1UKL, consistent with an elongated conformation of the complex. (B) Comparing the models in (B) with that in Fig S13B shows that the presence of a second Rev monomer in the specific antiparallel or C–C dimer arrangement examined yielded an improved or unchanged χ² value relative to that of the corresponding 1:1 Impβ/Rev complex, supporting the plausibility of these configurations for the 1:2 complex, whereas the specific transverse arrangement analyzed gave a poorer fit. However, the possibility that other transverse configurations might also yield a good fit cannot be excluded because only a small fraction of the 18,000 sterically allowed configurations for Rev monomer 2 were examined.

Figure S16.. Estimated concentrations of Impβ and Rev species as a function of total Rev concentration.

(A) Fractional occupancy of Rev binding sites 1 and 2 on Impβ. The intracellular concentration of Impβ, [Impβ]_cell, has been estimated at 1–2 μM (44) and is indicated by the black dashed line at 1.5 μM. The critical concentration, c_crit, above which Rev self-oligomerizes to form filaments in vitro has been reported to be 6 μM (103) and is indicated by a red dashed line. The concentration of Rev in virally infected cells varies over the course of infection, initially starting low and gradually rising as viral transcription proceeds (14). Because of the large value of [Impβ]_cell, only a small fraction of site 1 or site 2 is bound when Rev is in the low nM range, but this fraction rises quickly as Rev enters the high nM–low μM range. At subcritical concentrations, Rev can occupy up to 87% and 43% of sites 1 and 2, respectively. (B) Fraction of Impβ that is unbound (gray), in complex with one Rev monomer bound at either site 1 (blue) or site 2 (green) or in complex with two Rev monomers (black). A significant fraction (up to 38%) of Impβ can be bound simultaneously to two Rev monomers at subcritical Rev concentrations. (C) Fraction of Rev that is unbound (gray), bound to Impβ at site 1 (blue) or site 2 (green), or bound at either site (black). At low (nano- and sub-micromolar) Rev concentration, most of Rev (75%) is bound to Impβ, with 66% and 8% of all Rev molecules bound at sites 1 and 2, respectively. These fractions remain constant for concentrations below ∼0.2 and 1 μM, respectively. As the total Rev concentration rises above these levels, Impβ becomes progressively saturated with Rev and the fraction of unbound Rev increases correspondingly. (D) Fraction of total Impβ-bound Rev that is bound in a 1:1 complex at site 1 (blue), site 2 (green), or either site (white circles) or bound in a 1:2 complex (black). At Rev concentrations below ∼100 nM, the fractions of bound Rev that occupy either site 1 or site 2 in a 1:1 complex are ∼90% and 10%, respectively. As the Rev concentration rises above 100 nM, an increasingly significant fraction of bound Rev is located within an Impβ:Rev₂ complex, and this fraction can reach 57% before c_crit is attained. For panels (A, B, C, D), the concentrations of bound and unbound Impβ and Rev species were calculated using the equations K_d1 = (r − x − y) (b − x)/x and K_d2 = (r − x − y) (b − y)/y, where r is the total Rev concentration, x and y are the Rev concentrations bound to Impβ sites 1 and 2, respectively, b is [Impβ]_cell (taken as 1.5 μM), and K_d1 (0.61 μM) and K_d2 (5.3 μM) are the K_d values determined by Isothermal titration calorimetry for sites 1 and 2, respectively (Fig 4A). (A) The fractional occupancy (probability of the bound state), p₁ and p₂, of each binding site shown in (A) was given by p₁ = x/b and p₂ = y/b. (B) The fractions of Impβ species represented by the gray, blue, green, and black curves in (B) were calculated as the joint probabilities P_none = (1 − p₁) (1 − p₂), P_site1 = p₁(1 − p₂), P_site2 = (1 − p₁)p₂ and P_both = p₁p₂, respectively. (C) The fractions of total Rev represented by the gray, blue, green, and black curves in (C) were calculated as (r − x − y)/r, x/r, y/r and (x + y)/r, respectively. (D) The fractions of total Rev bound in an Impβ/Rev complex represented by the gray, blue, green, and black curves in (D) were calculated as (P_site1 + P_site2)/D, P_site1/D, P_site2/D and 2P_both/D, respectively, where D = P_site1 + P_site2 + 2P_both.

Figure S17.. Comparison of Impβ/Rev structural model with RanGTP- and cargo-bound complexes of Impβ.

(A) Rev bound to the Impβ C-site is predicted to overlap sterically with RanGTP. (B) Comparison with structures of cargo-bound Impβ complexes. (C) The Rev ARM motif is predicted to localize to the same volume as the N-terminal moiety of the Impα IBB domain.