The rates at which many genes are expressed as proteins are limited by the efficiency of a process called transcriptional elongation. This process takes place as the stretch of DNA that defines the gene is transcribed into an RNA molecule and it is catalyzed by an enzyme called RNA polymerase II. However, this enzyme can become trapped, and another enzyme called P-TEFb (positive transcription elongation factor b) is needed to release it. P-TEFb and other elongation factors therefore have an important role in gene expression.

The human immunodeficiency virus (HIV) is a retrovirus that hijacks the gene expression processes in human immune cells to replicate the RNA genome of the virus. To do this, the virus produces a protein called Tat that recruits P-TEFb as part of a multi-protein machine called the super elongation complex. This ensures that the process of transcriptional elongation, and hence the overall replication process, is highly efficient. There are gaps, however, in our knowledge of the architecture of the super elongation complex, which is known to be organized on a flexible scaffold. In turn, the molecular basis for the interaction between HIV-1 Tat and P-TEFb within the super elongation complex is not well understood.

Now Schulze-Gahmen et al. show that only one of the two subunits in P-TEFb—a cyclin known as CycT1—binds to the AFF4 scaffold protein in the super elongation complex. In addition to assisting with the expression of hundreds of human genes, super elongation complexes containing P-TEFb-AFF4 are hijacked in various forms of cancer and viral infections, including HIV/AIDS. Schulze-Gahmen et al. show that AFF4 can directly contact HIV-1 Tat, which binds to the P-TEFb-AFF4 complex much more strongly than it binds to P-TEFb alone. This suggests that HIV-1 Tat evolved to work within the super elongation complex. Moreover, Schulze-Gahmen et al. reveal that HIV-1 Tat binds to a cleft between the P-TEFb enzyme and the AFF4 protein, which raises the possibility that this cleft could be used as a target for anti-HIV/AIDS drugs.

At many genes in humans—including the integrated HIV genome as well as loci that regulate development and mediate responses to stress—RNA polymerase II initiates transcription but forms a stable paused complex after the synthesis of 30–50 nucleotides (Lin et al., 2011; Levine, 2012; Luo et al., 2012a; Zhou et al., 2012). These paused polymerases are poised for rapid, synchronous efficient transcription. For these genes, escape of the paused polymerase from the promoter-proximal region and elongation of the mRNA are rate-limiting regulated processes. Promoter escape requires positive transcription elongation factor b (P-TEFb), a heterodimeric protein kinase composed of CDK9 and cyclin T1 (CycT1) subunits. P-TEFb triggers promoter escape by directly or indirectly stimulating phosphorylation of the RNA polymerase II C-terminal domain and the associated factors, NELF (negative elongation factor) and DSIF (DRB sensitivity inducing factor). Consequently, recruitment of active P-TEFb to the paused polymerase complex serves as an important checkpoint for gene expression (Levine, 2012; Luo et al., 2012b; Zhou et al., 2012).

P-TEFb cycles between inactive and active complexes (Zhou and Yik, 2006). Recent studies of gene fusions in myeloid leukemias (Lin et al., 2010; Yokoyama et al., 2010), as well as complexes recruited to the HIV promoter by the HIV Tat protein (He et al., 2010; Sobhian et al., 2010; Jager et al., 2012), uncovered a family of Super Elongation Complexes (SECs) that bring together active P-TEFb and other transcription elongation factors. The SECs act at many normal human genes to stimulate mRNA elongation not only by triggering promoter escape but also by limiting proteolytic degradation of transcription elongation factors and increasing the processivity of RNAP II (He et al., 2010; Lin et al., 2010; Biswas et al., 2011; Liu et al., 2012). The SECs also couple to the PAF complex, which stimulates efficient transcript elongation (He et al., 2011). In addition to P-TEFb, the SECs contain subunits in the AF4, ELL, and ENL/AF9 protein families. Despite the major roles of SECs in metazoan gene expression and human disease, little is known about the architecture of these complexes.

To define the interactions that mediate SEC assembly and to understand how HIV Tat recruits P-TEFb (Wei et al., 1998) in the context of these large protein complexes, we mapped contacts among SEC subunits (Figure 1A) (Chou et al., 2012). Here, we report the structural and functional analysis of P-TEFb in complex with the cognate binding site on the SEC scaffold protein, AFF4. These studies reveal that AFF4 recognizes P-TEFb by binding the CycT1 subunit on the side opposite from CDK9. AFF4 is positioned to make direct contacts with HIV-1 Tat. Tat increases the affinity of P-TEFb for AFF4 by over an order of magnitude in vitro and rescues P-TEFb binding of AFF4 interface mutants in vivo. These results suggest that the SEC scaffold is an unanticipated direct partner of HIV-1 Tat, and an intersubunit Tat-binding pocket in the AFF4-P-TEFb complex may afford an unexpected site to target with selective inhibitors of HIV transcription.

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The AF4 proteins, AFF1–4, form intrinsically disordered scaffolds that bind other transcription elongation factors not through folded domains but rather through dispersed short binding sites in the first 750 amino acids (Chou et al., 2012; Leach et al., 2013) (Figure 1A). Residues 2–73 of AFF4, for example, are sufficient to bind P-TEFb through the CycT1 subunit, and a peptide encompassing AFF4_2–73 folds upon binding CycT1. Using this binding site, we determined the 2.9-Å-resolution crystal structure of AFF4_2–73-P-TEFb-AMPPNP (R/R_free = 0.207/0.245; Figure 1, Figure 1—figure supplements 1 and 2, and Table 1).

In all three independent copies of the complex in the crystals, AFF4 residues Leu34-Ile66 are ordered, binding to the second cyclin domain of CycT1 opposite CDK9 (Figures 1B, 2). In one complex, an isolated helix containing AFF4 residues 3–21 bridges symmetry-related CDK9 molecules in the crystals (Figure 1—figure supplement 2). We focus on the shared features of the three independent complexes. Consistent with the coupling of folding to binding, AFF4 lacks intramolecular tertiary contacts as it snakes across the CycT1 surface. An extended segment in AFF4 tracks antiparallel to the H3′-H4′ loop in CycT1, and two short AFF4 helices fit into shallow grooves in CycT1 between H3′ and H5′ and the surface formed by H2′ and H3′ (Figure 2A). Compared to the separated models, 1457 Ų of AFF4 and 1251 Ų of CycT1 accessible surface are buried in the complex. Twenty-six of the 33 ordered residues in AFF4 contact CycT1, emphasizing the extensive recognition determinants in the scaffold.

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Many hydrophobic and aromatic residues in CycT1 mediate contacts with AFF4 (Figure 2B–C and Figure 2—figure supplement 1). A pocket between CycT1 Leu163-Val164-Arg165, Trp221, and Tyr224, for example, anchors AFF4 Phe35, which forms a classic edge-to-face interaction (Burley and Petsko, 1985) with the Tyr (Figure 2C). CycT1 Trp210 adjusts to create a site that buries AFF4 Pro38 and allows a short antiparallel β-sheet to form between AFF4 Tyr39-Val41 and CycT1 Asn 209-Glu211 (Figure 2—figure supplement 1A). CycT1 Trp207 shifts to form a hydrogen bond with the Gly57 carbonyl in the loop between the two AFF4 helices, additional van der Waals contacts with the loop backbone and nonpolar contacts with AFF4 residues Leu56 and Tyr59 (Figure 2—figure supplement 1B). Eight intermolecular hydrogen bonds help establish the chemical complementarity between AFF4 and CycT1. Twenty-one of the 26 interacting residues in AFF4 are conserved in AFF1–3 (Figure 2—figure supplement 2).

In comparison to the structure of P-TEFb alone (CDK9_1–330/CycT1_1–259; PDB ID 3BLQ; Baumli et al., 2008), the last 20 residues present in the CycT1 subunit undergo a major rearrangement upon AFF4 binding (Figure 2D). AFF4 overlaps with the position of residues 243–259 in the superimposed P-TEFb cyclin subunit. To accommodate AFF4, helix H5′ straightens and residues 251–256 form a helical segment that packs against CycT1 helices H1 and H2' and forms part of the AFF4 binding surface. In addition to burying hydrophobic residues in the first AFF4 helix, the adjustments in CycT1 helix H5′ mediate formation of a hydrogen-bonded ion pair between AFF4 Arg51 and CycT1 Glu246. CycT1 Trp256, the last residue ordered in the AFF4 complex, moves over 13 Å upon AFF4 binding.

To probe the basis for CycT1 binding, we measured the effects of interface mutations on the stability of the AFF4 complex in vitro. The AFF4 2–363 and 33–67 fragments bound to CycT1 with similar affinities (Table 2), suggesting that the ordered AFF4 segment in the crystal structure captures the major CycT1 binding determinants. Mutations throughout the CycT1 interaction surface reduced AFF4 binding (Figure 3A and Table 3). The Trp210Ala and Trp207Ala substitutions in CycT1 had the largest effects, respectively, reducing AFF4 affinity by 21- and 58-fold. These results point to these CycT1 Trp residues as interaction hotspots and suggest that contacts all along the interface observed in the crystal structure mediate AFF4 recognition.

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To examine the functional roles of the N-terminal 72 residues of AFF4, we measured the effects of tandem alanine mutations on the stimulation of expression of a luciferase reporter gene driven by the HIV-1 promoter in HeLa cells (He et al., 2011) (Figure 3B and Figure 3—figure supplement 1). Consistent with the structure, mutations of AFF4 residues that contact P-TEFb reduced Tat-independent transcriptional stimulation. Three of the most deleterious variations—alanine substitutions at Pro33/Leu34, Phe35 (Ala36 was maintained), and Met55/Leu56—shorten hydrophobic side chains that are buried in the interface. Tandem alanine substitutions (Glu45/Asp46 and Gly57/Asn58) that remove side chains that cap and stabilize the AFF4 helices also reduced luciferase expression. In contrast, residues such as Glu61/Met62 and Ile71/Pro72, which are more tolerant of di-alanine substitutions, are more solvent exposed or flexible. Tandem alanine replacements in residues 3–32, which flank the ordered CycT1 contacts in the crystal structure, showed significant but generally smaller effects on AFF4 transcriptional stimulation activity (Figure 3B). These results implicate the P-TEFb binding site, as well as the flanking flexible sequences, in the function of the AFF4 N-terminal segment.

The HIV-1 Tat protein was shown nearly 15 years ago to bridge P-TEFb and the TAR RNA element near the 5′ end of nascent HIV transcripts to recruit the active CDK9 kinase to the HIV promoter (Wei et al., 1998). The discovery that Tat recruits P-TEFb as part of a larger SEC (He et al., 2010; Sobhian et al., 2010) raised the question of how Tat distinguishes the SEC from free P-TEFb, particularly since Tat binds P-TEFb in isolation. Moreover, Tat shows specificity for SECs containing AFF4 and AFF1 (He et al., 2010; Sobhian et al., 2010). What accounts for this specificity? The AFF4-P-TEFb crystal structure provides a simple and unsuspected explanation—Tat binds in a position to make direct contacts with the scaffold (Figure 4A). Superposition of the CycT1 subunits of the structures of Tat-P-TEFb (Tahirov et al., 2010) and AFF4-P-TEFb shows that Tat is positioned to pack against helix 2 of AFF4. Tat Met1, Lys28 (which is reversibly acetylated in vivo; Kiernan et al., 1999; Ott et al., 2011) and Phe32, in particular, are predicted to interact with AFF4 Glu61, Met62, Phe65, and Ile66. The disordered C-terminus of the AFF4 peptide also neighbors Glu2 and His13-Gly15 of Tat. These seven residues of Tat are crucial for transcriptional activation, even though they are exposed to solvent in the Tat-P-TEFb complex (D'Orso et al., 2012).

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The juxtaposition of AFF4 and Tat on the P-TEFb surface predicts that the scaffold enhances Tat binding. Direct measurements of Tat affinity are problematic, however, because Tat is unstable in vitro in the absence of partners and difficult to maintain in an active form. To overcome this problem, we took advantage of a thermodynamic cycle that illustrates that AFF4 and Tat mutually influence the P-TEFb affinity of each other by the same amount (Figure 4—figure supplement 1). In quantitative in vitro assays (Figure 4B and Table 2), the purified AFF4_2–73 peptide bound Tat-P-TEFb (K_d = 0.85 ± 0.15 nM) ∼11 times more tightly than P-TEFb (K_d = 10 ± 1 nM). The AFF4_2–363 segment also bound Tat-P-TEFb (K_d = 0.6 ± 0.1 nM) ∼11 times more tightly than P-TEFb (K_d = 7 ± 1 nM). These results support the model of direct Tat interactions with AFF4 and suggest that the AFF4_2–73 peptide includes the principal residues that contact Tat (Figure 4 and Figure 4—figure supplement 1).

To test the importance of the AFF4 interactions for P-TEFb and Tat recognition in vivo, we measured the effects of mutations in AFF4 on the binding of SEC subunits and Tat in HeLa cells. Cells were transfected with wild-type or mutant AFF4 containing a C-terminal 3× FLAG tag, and immunoprecipitations using an anti-FLAG antibody were probed for the presence of associated factors. Alanine substitutions in P-TEFb contacts such as AFF4 Phe35, Tyr59/Asp60, and Lys63/Asp64 reduced binding of P-TEFb but not the ELL2, AF9, or ENL subunits of the SEC (Figure 4C). These defects were rescued by overexpressing a stably integrated gene for Tat, which strengthened the AFF4-P-TEFb association. In contrast, tandem alanine substitutions for AFF4 Glu61/Met62, which are more exposed in the P-TEFb interface and predicted to contact Tat, caused a small reduction (∼30%) in the binding of P-TEFb that was not rescued by overexpressing Tat (Figure 4C). The specificity of these mutational effects supports the model that Tat interacts directly with AFF4.

In keeping with the tenuous contacts of the AFF4_3–21 helix with CDK9, AFF4_2–73 (K_d = 10 ± 1 nM) binds only 3.6 times more tightly than AFF4 _32–67 (K_d = 36 ± 4 nM) to P-TEFb (Table 2). In addition, the CDK9 kinase subunit structure is little changed upon AFF4 binding to P-TEFb (CDK9_15–360 RMSD = 0.54 Å vs 1BLQ). In vivo, the Glu13Ala/Arg14Ala AFF4 mutant in the heart of the CDK9 interface in the crystals is associated with a <25% decrease in transcription stimulation (Figure 3B) and little change in P-TEFb binding (Figure 4C). To measure if recruitment of P-TEFb to the SEC scaffold regulates the kinase, we assayed the effects of AFF4 on the in vitro phosphorylation of an RNA polymerase II CTD substrate by purified P-TEFb and Tat-P-TEFb (Bitoun et al., 2007). AFF4_2–73 inhibited CTD phosphorylation by approximately twofold in a purified system (Figure 5 and Figure 5—figure supplements 1 and 2). By comparison, Tat stimulated P-TEFb by sevenfold, and addition of AFF_2-73 did not further stimulate the kinase activity of the Tat-P-TEFb complex. Taken together, these results point away from CDK9 as the primary physiological partner of AFF4_3–21 and suggest that AFF4 functions as a SEC scaffold but not an allosteric activator of CDK9.

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The AFF4-P-TEFb crystal structure reveals that a high density of contacts in residues 34–66 of the AFF4 scaffold mediates binding to the CycT1 subunit of P-TEFb. These contacts are largely conserved in AF4 family members and can be perturbed physically and functionally by mutations (Figure 3 and Figure 2—figure supplement 2). Consistent with these results, tandem alanine substitutions of Pro33/Leu34, Val41/Thr42, Arg51/Ile52, and Met55/Leu56 in the CycT1 binding site (but not Arg3/Glu4 or Glu25/Asp26 in the preceding segment) also reduce P-TEFb binding in vivo (Chou et al., 2012). The impacts of alanine substitutions on transcriptional stimulation by AFF4 (Figure 3B) show that residues in the P-TEFb interface, as well as helix stabilizing residues, play crucial roles. The additional sensitivity of transcriptional stimulation to alanine substitutions of disordered residues flanking the CycT1 binding site suggests that the flexibility and potential interactions with other ligands are important for function.

HIV-1 Tat binds adjacent to AFF4, increases the affinity of AFF4 for P-TEFb by over an order of magnitude, and rescues P-TEFb binding to AFF4 interface mutants in vivo. The AFF4 Glu61/Met62 double alanine substitution in the proposed Tat interface blocks this rescue. These results suggest that direct contacts between Tat and the AFF4 scaffold in complex with P-TEFb mediate the selective recruitment of the SEC to the HIV promoter. The functional AFF4-Tat interface, including the acetylated Lys28 in Tat as a potential regulator of affinity (Kiernan et al., 1999), supports the idea that Tat evolved to function within the SEC. In the crystal structure of the (low-affinity) Tat-P-TEFb subcomplex (Tahirov et al., 2010), Tat binds to a relatively open groove. In the context of the AFF4-P-TEFb structure, however, AFF4 creates an unanticipated pocket for Tat (Figure 4D). This pocket may ultimately provide a suitable therapeutic target for the development of small-molecule inhibitors of Tat binding that selectively block HIV transcription.

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Author details

1. Ursula Schulze-Gahmen

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   US-G, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Heather Upton

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   HU, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Andrew Birnberg

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   AB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Katherine Bao

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Present address

   Department of Immunology, Duke University School of Medicine, Durham, United States

   Contribution

   KB, Conception and design, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

5. Seemay Chou

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Present address

   Department of Microbiology, University of Washington, Seattle, United States

   Contribution

   SC, Conception and design, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

6. Nevan J Krogan

   1. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
   2. California Institute for Quantitative Biosciences, QB3, Berkeley, United States
   3. J David Gladstone Institutes, San Francisco, United States

   Contribution

   NJK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Qiang Zhou

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   QZ, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

8. Tom Alber

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. California Institute for Quantitative Biosciences, QB3, Berkeley, United States

   Contribution

   TA, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   tom@ucxray.berkeley.edu

   Competing interests

   The authors declare that no competing interests exist.

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