AFF4 binding to Tat-P-TEFb indirectly stimulates TAR recognition of super elongation complexes at the HIV promoter
- University of California, Berkeley, United States
Figures
Figure 1 with 3 supplements
Tat and AFF4 bind adjacent to each other on the CycT1 surface.
(A) Tat-AFF4-P-TEFb ribbon diagram (left) showing interactions between Tat (red) and AFF4 (blue) bound to the CycT1 (yellow) subunit of P-TEFb. AFF4 helix 0 is bound to the CDK9 (cyan) subunit, and …
Figure 1—figure supplement 1
Crystal contacts of CycT1 TRM for two representative complexes in the a.u..
(A) In the two dyad-related complexes, CDK9 (gray sticks) molecules from adjacent complexes make contacts with the C-terminal end of the CycT1 (yellow) TRM, as well as AFF4 (blue). (B) In the third …
Figure 1—figure supplement 2
Surface representation of the binding pocket for Tat M1 and the N-acetyl group.
The methionine side chain binds in a pocket formed by Tat (red) and CycT1 (yellow).
Figure 1—figure supplement 3
Schematic drawing of AFF4 secondary structures.
Disordered regions (gray rectangles), α helices (blue springs) and the short β strand (black arrow) are indicated. Helix H0 in AFF4 is only observed in two out of three molecules in the a.u.
Figure 2
Structural shifts in the subunits of the Tat-AFF4-P-TEFb complex.
(A) Superposition of the AFF4-P-TEFb complex (PDB ID 4IMY, pastel colors) and Tat-AFF4-P-TEFb (red, blue, yellow) on the CycT1 subunit shows coupled shifts of the two AFF4 helices. AFF4 helices 1 …
Figure 3 with 1 supplement
CycT1 TRM interacts with Tat and AFF4.
(A) Ribbon diagram of two distinct TRM conformations observed in the Tat-AFF4-P-TEFb crystal structure (red, blue, yellow/dark red, light blue, green). Zn2+ ions are shown as gray spheres. (B) …
Figure 3—figure supplement 1
Representative electron density for the Tat-AFF4-P-TEFb complex.
2Fo-Fc map (1.0 σ) for Tat (red) and CycT1 TRM (green) is shown for a dyad-related complex. Residues of the CycT1 TRM were omitted from the model used for molecular replacement and subsequently …
Figure 4
AFF1 Tat interaction mutants reduce Tat binding and activation of HIV LTR by AFF1.
(A) Nuclear extracts (NE) were prepared from HeLa cells expressing the truncated Flag-tagged AFF1 protein (residues 1–308). Anti-Flag immunoprecipitates (IP) from the NE were examined by Western …
Figure 5 with 1 supplement
SECs stimulate TAR recognition.
(A) Electrophoretic mobility shift assays with 32P-labeled TAR and increasing concentrations of Tat-P-TEFb, or Tat-P-TEFb + AFF432–67, Tat-P-TEFb + AFF42–73, Tat-P-TEFb + AFF42–98. Control assays …
Figure 5—figure supplement 1
Model of TAR binding to SEC.
The positively charged CycT1 TRM is positioned close to the predicted location of the Tat ARM, which binds to bases in the TAR bulge (U23–U25) (Weeks and Crothers, 1991). Considering that the CycT1 …
Tables
Table 1
X-ray data collection and refinement statistics for P-TEFb-Tat-AFF4
| Data collection | |
| Space group | P6522 |
| Cell dimensions: a, b, c | 184.91, 184.91, 360.40 |
| Resolution (Å)* | 50.0–3.0 (3.05–3.0) |
| Unique reflections* | 73,424 (3589) |
| I/σ(I)* | 12.8 (0.9) |
| Rmerge (%)* | 22.2 (>100) |
| Rmerge (%)*, I/sigI≥3 | 8.4 (18.9) |
| Rpim (%)† | 7.6 (87.9) |
| CC1/2 high resolution shell | 0.553 |
| Completeness (%)* | 100.0 (100.0) |
| Redundancy* | 24.2 (23.8) |
| Temperature (K) | 100 |
| Mosaicity (°) | 0.23–0.39 |
| Refinement | |
| Resolution (Å) | 49.0–3.0 |
| No. reflections | 73,297 |
| Rwork/Rfree* | 0.206/0.232 (0.316/0.335) |
| No. atoms/B-factors (Å2) | |
| CDK9, molecule 1, 2, 3 | 2560 (75.4), 2521 (90.9), 2572 (88.5) |
| Cyclin T1, molecule 1, 2, 3 | 2061 (79.4), 2053 (85.8), 2058 (97.8) |
| AFF434-66, molecule 1, 2, 3 | 438 (85.0), 268 (115.7), 422 (92.3) |
| Tat | 390 (79.1), 384 (78.0), 390 (102.7) |
| Water | 37 (58.7) |
| R.m.s. deviations | |
| Bond lengths (Å) | 0.0035 |
| Bond angles (°) | 0.811 |
| Ramachandran plot‡ | |
| Favored (%) | 96.0 |
| Allowed (%) | 3.36 |
| Disallowed (%) | 0.66 |
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*
Values in parentheses are for the highest resolution shell.
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†
Rp.i.m. is the precision-indicating merging R factor, which is related to the traditional Rsym but provides a better estimate of data quality (Weiss and Hilgenfeld, 1997; Weiss et al., 1998).
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‡
Values from MOLPROBITY (Chen et al., 2009).
