Figure 4. AFF4 binds in position to make direct contacts with HIV-1 Tat.
(A) Superposition of the AFF4-P-TEFb complex and the Tat-P-TEFb complex using the cyclin subunit (yellow) shows the close proximity of AFF4 (blue) and Tat (red). Tat Lys28 (pink), where acetylation stimulates function, as well as other residues essential for Tat transcriptional activation (D'Orso et al., 2012) that are exposed to solvent in the Tat-P-TEFb complex (bright red) are positioned adjacent to AFF4. (B) Tat enhances AFF4 binding in vitro. Fluorescence polarization of fluorescein-labeled AFF432–67 (5 nM) is plotted as a function of the concentration of CycT1 (blue circles), P-TEFb (red squares), and Tat-P-TEFb (green triangles). (C) Alanine substitutions in the P-TEFb binding site of AFF4 reduce CycT1 binding but not associations of other SEC subunits in HeLa cells. Western blots show associations of each indicated factor with different FLAG-tagged AFF4 variants (top). Lysates were immunoprecipitated with an anti-FLAG antibody. Expression of Tat (right) rescues defects in CycT1 binding, except for the E61/M62 double alanine mutant. This mutant in the predicted AFF4-Tat interface shows equal small defects in P-TEFb binding in the absence (left) and presence (right) of Tat. (D) AFF4 (blue) and CycT1 (yellow) create an intersubunit pocket where Tat (red) can bind with minor structural adjustments. The program DoGSiteScorer (Volkamer et al., 2012) assigns this cleft a high druggability score (0.83 out of 0–1.0) and shows that it contains the most nonpolar surface of any pocket in the AFF4-P-TEFb structure.
Figure 4—figure supplement 1. Thermodynamic cycle for AFF4 and Tat binding to P-TEFb.
