The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat
- University of California, Berkeley, United States
- University of California, San Francisco, United States
- California Institute for Quantitative Biosciences, QB3, United States
- J David Gladstone Institutes, United States
Figures
Figure 1 with 2 supplements
AFF4 binds CycT1 distal to CDK9.
(A) Schematic model of the SEC. AFF4 is an intrinsically disordered scaffold that binds partners via 20–50 residue segments. (B) Ribbon diagram showing the strand–helix–helix arrangement of AFF4 …
Figure 1—figure supplement 1
Electron density for AFF42–73.
Fo-Fc omit map (3.5 σ, gray) for residues 34–66 of AFF4. Anomalous difference map (5 σ, red) shows the positions of the methionine residues in AFF4. The anomalous difference Fourier was calculated …
Figure 1—figure supplement 2
A crystal contact formed by AFF42–21.
(A) An isolated helix from the aff4 N-terminus, packs loosely against αE and αI of one CDK9 subunit (chain C) and makes contacts with the β2-β3 loop of a symmetry-related CDK9 molecule. (B) …
Figure 2 with 2 supplements
Basis for AFF4 scaffold recognition by P-TEFb.
(A) AFF4 residues 34–66 (blue spheres) fill grooves on CycT1 (yellow surface). (B) Chemical complementarity mediates AFF4 binding. Exposed hydrophobic residues of CycT1 (yellow surface) are buried …
Figure 2—figure supplement 1
Example interactions between AFF4 and P-TEFb.
(A) Main-chain hydrogen bonds between the extended aff4 peptide 34–41 and CycT1. (B) Hydrophobic interactions with CycT1 Trp207 and hydrogen bonds of CycT1 Trp207 and Asp169 anchor the aff4 α1–α2 …
Figure 2—figure supplement 2
Conserved AFF4 sequences mediate P-TEFb recognition.
Multiple sequence alignment of amino acids 1–78 (AFF4 numbering) for human and mouse AF4 family members.
Figure 3 with 1 supplement
AFF4 interface mediates P-TEFb recognition.
(A) Mutations of CycT1 contact residues reduce AFF4 affinity. Fluorescence polarization of fluorescein-labeled AFF432–67 (5 nM) is plotted as a function of the concentration of the indicated CycT1 …
Figure 3—figure supplement 1
Expression levels of AFF4 variants.
(A) Representative Western blot probed with anti-FLAG antibodies to measure the level of FLAG-tagged aff4 variants in HeLa cell lysates. (B) Expression levels of aff4 variants. Values are the …
Figure 4 with 1 supplement
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 …
Figure 4—figure supplement 1
Thermodynamic cycle for AFF4 and Tat binding to P-TEFb.
The enhancement of Tat affinity for P-TEFb by aff4 is equal to the ratio of aff4 affinity for P-TEFb in the presence and absence of Tat.
Figure 5 with 2 supplements
Kinase activity of P-TEFb and P-TEFb-Tat complexes with AFF4.
(A) Autoradiogram showing phosphorylation of GST-CTD (500 ng) by P-TEFb and P-TEFb-Tat with and without excess (0.28 μM) AFF42-73 in the presence of low (50 μM) ATP. (B) Phosphorylation of 500 ng …
Figure 5—figure supplement 1
SDS polyacrylamide gel of P-TEFb and P-TEFb-Tat at the same ratio as they were used in the kinase assay.
https://doi.org/10.7554/eLife.00327.017
Figure 5—figure supplement 2
Western blots of kinase reaction products from panel B. Phosphorylated CTD was detected with anti-phoshoSer2 and anti-phoshoSer5 antibodies.
The GST-CTD was phosphorylated on Ser2 and Ser5. However, the Ser2 phosphorylation was detected only on the full-length CTD, while Ser5 phosphorylation was detected disproportionately on proteolytic …
Tables
Table 1
X-ray data collection and refinement statistics for AFF4-P-TEFb-AMPPNP
| Data collection | AFF4-P-TEFb-AMPPNP |
| Space group | P212121 |
| Cell dimensions: a, b, c | 100.691, 126.298, 195.626 |
| Resolution (Å)* | 50-2.94 (2.99–2.94) |
| Unique reflections* | 54,189 (2664) |
| Rsym (%)* | 9.3 (>100) |
| I/σ(I)* | 23.2 (1.3) |
| Completeness (%)* | 100.0 (100.0) |
| Redundancy* | 8.1 (7.5) |
| Temperature (K) | 100 |
| Mosaicity (°) | 0.45–0.6 |
| Refinement | |
| Resolution (Å) | 48.7-2.94 |
| No. reflections | 53,775 |
| Rwork/Rfree | 0.207/0.245 |
| No. atoms/B-factors (Å2) | |
| CDK9, molecule 1, 2, 3 | 2558 (111.9), 2533 (116.3), 2558 (121.6) |
| Cyclin T1, molecule 1, 2, 3 | 2003 (121.3), 2024 (123.1), 2001 (118.5) |
| AFF4, molecule 1, 2, 3 | 248 (156.3), 421 (161.1), 243 (160.3) |
| Water | 19 (90.1) |
| Root mean square deviations | |
| Bond lengths (Å) | 0.004 |
| Bond angles (°) | 0.666 |
| Ramachandran plot† | |
| Favored (%) | 94.7 |
| Allowed (%) | 4.48 |
| Disallowed (%) | 0.78 |
| Protein Data Bank ID | 4IMY |
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*
Values in parentheses are for the highest resolution shell.
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†
Values from MOLPROBITY.
Table 2
Binding affinities of AFF4 segments
| Direct binding | Competition assay | |||
| AFF432–67 | AFF432–67 | AFF42–73 | AFF42–363 | |
| CycT1 | 104 ± 17 nM | 102 ± 10 nM | 130 ± 18 nM | 115 ± 15 nM |
| P-TEFb | 36 ± 6 nM | 36 ± 4 nM | 10 ± 1 nM | 7 ± 1 nM |
| Tat-P-TEFb | 8.8 ± 0.8 nM | 4.5 ± 0.6 nM | 0.85 ± 0.15 nM | 0.6 ± 0.1 nM |
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Dissociation constants measured by direct binding of fluorescein-labeled AFF432–67 and by competition with unlabeled AFF4 segments. The increased affinity of AFF4 for P-TEFb compared to CycT1 may be due to structural changes in the cyclin subunit or additional interactions with the CDK9 kinase subunit. The similar affinities of AFF42–73 and AFF42–363 for all the cyclin-containing species suggest that AFF42–73 encompasses the binding sites for P-TEFb and Tat-P-TEFb.
Table 3
Dissociation constants of AFF432–67 for Cyclin T1 mutants
| CycT1 variant | Kd (nM) |
| Wild-type | 104 ± 17 |
| Y175A | 228 ± 18 |
| E211A | 356 ± 29 |
| D169A | 438 ± 41 |
| F176A | 645 ± 58 |
| R165A | 1592 ± 171 |
| W210A | 2190 ± 246 |
| W207A | 6050 ± 871 |
