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How the AFF1/4 scaffold recruits the elongation factor ELL2 to promote HIV-1 proviral transcription

The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the superelongation complex required for HIV-1 proviral transcription. We determined the 2.0-Å resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4-ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.


Introduction
Curing Acquired Immunodeficiency Syndrome (AIDS) is a major global health goal.
AIDS is caused by the human immunodeficiency virus (HIV), which has proved exceptionally difficult to eradicate . The principal obstacle to HIV eradication is the persistence in patients of a reservoir of cells harboring latent provirus integrated within the genome (Ruelas and Greene, 2013). Clinical interest in the reactivation of latent HIV  has brought renewed attention to the mechanism of transcriptional regulation of the HIV provirus. Latency is regulated at the levels of epigenetic silencing and transcription initiation and elongation (Mbonye and Karn, 2014). Transcription elongation, which is promoted by the HIV Tat protein and TAR RNA sequence, is the best-understood of these mechanisms. The functions of HIV Tat and TAR in promoting elongation are completely dependent on their ability to hijack the host Super Elongation Complex (SEC) (He et al., 2010;Lu et al., 2014;Sobhian et al., 2010).
The SEC consists of the Cyclin-dependent kinase CDK9 and Cyclin T (CycT1 or T2), together known as P-TEFb (Price, 2000); one of either of the intrinsically disordered (ID) scaffold proteins AFF1 or AFF4 (He et al., 2010;Sobhian et al., 2010); one of either ENL or AF9; and one of either of the RNA polymerase elongation factors ELL1 or ELL2 (Biswas et al., 2011;He et al., 2010;Luo et al., 2012). The reason that Tat is such a powerful activator of HIV-1 transcription lies in its ability to pack two distinct transcriptional elongation factors P-TEFb and ELL1/2 into a single SEC complex, where the two factors can synergistically stimulate a single RNA Pol II elongation complex (He et al., 2010;Lu et al., 2014). AFF1/4 is more than 1100 residues long and is the principal scaffold that holds the SEC together (Lin et al., 2010). AFF1/4 consists almost entirely of predicted intrinsically disordered regions (IDRs). AFF1 and AFF4 function in transcription elongation by virtue of various peptide motifs interspersed throughout their sequences, much like many other ID signaling and regulatory proteins that have come under intensive study (Csizmok et al., 2016;Tompa et al., 2014). The AFF1-and ELL2containing version of the SEC is the most important in the promotion of proviral elongation, despite its low abundance (Li et al., 2016).
The structure of P-TEFb lacking the C-terminal IDR of CycT1 has been determined in complex with HIV-1 Tat (Tahirov et al., 2010) and the N-terminal 60 residues of AFF4 (Gu et al., 2014;. This structure shows that AFF4 residues 32-67 bind as an extended strand followed by two α-helices to the CycT1 surface. NMR studies showed that AFF4 residues 761-774 fold into a β-strand that combines with two strands of the AF9 AFF4-binding domain to generate a threestranded β-sheet (Leach et al., 2013). The structures of the P-TEFb and AF9 complexes with AFF4 revealed two of the three known interfaces used by AFF4 in assembly of the SEC. In this study, we set out to visualize the last of the three known interfaces critical for AFF4 function, its binding site for ELL1/2. Progress in characterizing the AFF4 interface with ELL2 has been slower than for the P-TEFb and ENL/AF9 interfaces, in part because the AFF4 binding domain for ELL2 is poorly soluble and prone to aggregation. The first step in this study was to obtain a fusion construct such that a stable obligate complex between ELL2 and AFF4 was formed. This fusion-based tethered complex was stable and soluble enough to be crystallized. The crystal structure confirmed that the AFF4 binding domain of ELL2 has an occludin fold, as predicted from sequence homology (Li et al., 2005). It showed that the IDR consisting of AFF4 residues 301-351 (hereafter referred to as AFF4 ELLBow for ELL1/2 Binding) folds up into a helix and elbow joint arrangement that makes extensive contacts with the occludin domain of ELL2 (hereafter ELL2 Occ ). These results complete the structural picture of how AFF1/4 engages its three known partners in the SEC.

Mapping the AFF4 ELLBow and ELL2 Occ interaction
Following the initial mapping of the AFF4 and ELL2 interaction sites to approximately residues 318-337 of the former and 519-640 of the latter   (Fig. 1A), we sought to isolate a stable form of this monomeric ( Fig. 1-S1A) complex for crystallization.
Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to probe the protection of expected helical regions of ELL2 Occ in variant constructs consisting of AFF4 301-351 -(Gly-Ser) 4 -ELL2 519-640 and AFF4 312-330 -ELL2 519-640 . Essentially complete peptide coverage was obtained for both constructs (Fig. 1B). The longer construct manifested some exchange-protected regions (330-332) absent in the shorter construct (see first dashed box above and below in Fig. 1B). The data for the longer construct also showed that the ELL2 sequence 540-554, corresponding to part of the first predicted helix (second dashed box above and below in Fig. 1B), manifested < 10 % exchange over 10 s. In comparison, a higher level of exchange was observed in the shorter construct. We proceeded to focus on crystallizing AFF4 301-351 -(Gly-Ser) 4 -ELL2 519-640 .
Helices α2 (584-602) and α3 (607-638) of ELL2 are oriented at an angle of ~100° with respect to each other such that both pack along the length of the long, bent helix α1 (Fig.   2B). The structure confirms that ELL2 Occ has the same arch-shaped three-helix fold as the C-terminal domain of occludin (Li et al., 2005). The ELL2 Occ and occludin C-terminal domain (pdb entry 1XAW) structures can be superimposed with an r.m.s.d. of 4.0 Å for 104 residue pairs (Fig. 2C). The main differences are in the α2-α3 connector and in the mutual orientation of these two helices. The α2-α3 angle is steeper in ELL2 Occ than in occludin. A minor difference is that ELL2 Occ has an extra single-turn helix, denoted α0, at its N-terminus. AFF4 ELLBow is ordered over residues 314-349 and buries 1535 Å 2 of solventaccessible surface area. Fully 37% of the entire solvent accessible surface area of AFF4 ELLBow is buried in the contact. The AFF4 ELLBow sequence folds into several distinct regions. It begins with helix α1 (315-324), is followed by an extended hydrophobic sequence (325-327), a polyproline segment (328-330), an extended region that doubles back on itself in what we refer to as the ELLBow joint (331-343), and a second extended hydrophobic sequence (344-349) (Fig. 3A). The fusion construct contains 17 residues that are not visualized in electron density, AFF4 351-351, 8 Gly-Ser linker residues, and ELL2 519-524, more than adequate to span the 15 Å between AFF4 349 and ELL2 525 in the structure. Hydrophobic side-chains of AFF4 ELLBow α1, including Val316, Ile319, Leu320, and Met323, are buried in a hydrophobic groove formed by the C-terminal half of ELL2 Occ-α1 and α2 (Fig. 3B). These helices of ELL2 Occ contribute hydrophobic residues Val565, Phe569, Ile570, Leu572, Aps573, Val589, His590, Tyr596, Leu594 and Ile599 to the AFF4 α1 binding site (Fig. 3B). ELL2 Occ buries 1315 Å 2 of solventaccessible surface area, corresponding to 15% of its total surface area.
AFF4 ELLBow is centered on Trp327, which forms extensive hydrophobic interactions with the side-chains of ELL2 residues His 559, Met562, Cys614 and Glu615.
The Trp327 indole nitrogen also forms a water-mediated hydrogen bond with the Tyr607 hydroxyl. This cluster of residues is completed by the side chains of AFF4 Pro328, Phe345 and Phe347 (Fig. 3C). Collectively, this cluster forms an extensive interaction network in which AFF4 ELLBow folds up not only against ELL2 but also against itself.
In the AFF4 ELLBow joint, the side chain of Leu331 sticks into a pocket comprising Tyr 552, Tyr555, His618, Leu621 and Ala622 of the N-terminal half of ELL2 Occ-α1 and α3. The side chain of Ile334 packs against the side chains of Lys545, Phe547, Lys625 and Leu628. At the distal end of ELLBow joint, Pro342 falls into a shallow cavity composed of Ala622, Lys625 and Arg626 (Fig. 3D).
A number of hydrogen bonds are observed in the complex (Fig. 4A). In AFF4 ELLBow α1, the side-chains of Asp317 and Arg576 of ELL2 form a bidentate salt-bridge with one another (Fig. 4B). Glu322 of AFF4 forms a 2.8 Å salt bridge with one of the two observed rotamers of His608 of ELL2 (Fig. 4B). In the central cluster, the carbonyl group of Pro328 forms a 2.7 Å hydrogen bond with the side chain of His559 of ELL2 ( Fig. 4C). Moving into the ELLBow joint, the main chain amide and carbonyl of AFF4 Leu331 form hydrogen bonds with the hydroxyl oxygens of Tyr552 and Tyr555, respectively, of ELL2. A 2.6 Å hydrogen bond is formed between Thr332 of AFF4 and Lys625 of ELL2 (Fig. 4D). The Ile334 carbonyl accepts a hydrogen bond from the sidechain of Lys545. The Cys338 main-chain amide donates a hydrogen bond to the sidechain of Asp632. The main-chain amide of Phe345 forms a 2.9 Å hydrogen bond with the side chain of Gln619 (Fig. 4D).
The AFF4 ELLBow :ELL2 Occ complex was screened for cavities using POCASA 1.1 (POcket-CAvity Search Application) (Yu et al., 2010) with a probe radius of 3 Å. Of the five largest cavities located, one of these is an internal cavity at the AFF4 ELLBow :ELL2 Occ interface (Fig. 5A). The cavity is 36 Å 3 in volume and is connected to the exterior by a narrow mouth (Fig. 5B). It is lined by the aliphatic part of Glu322, Met323, His325, Trp327, Phe347, and Pro348 of AFF4 and by Met562, Ala566, Tyr607, and the aliphatic part of Lys611 of ELL2 (Fig. 5C). These residues are in or adjoin the central cluster part of the interface.

Function of the AFF4 ELLBow :ELL2 Occ interface in binding
To validate whether that the observed structural interface corresponded to the mode of binding of AFF4 ELLBow and ELL2 Occ in solution, we carried out a series of mutant peptide binding assays using fluorescence polarization. We considered this particularly critical given the use of the fusion construct to obtain crystals. The assay monitored the displacement of fluorescently labeled wild-type ELLBow peptide by unlabeled mutant peptides 301-351. The unlabeled wild-type peptide in this system has K d = 86 nM (Table   2; Fig. 6A). The AFF4 hydrophobic residues Val316, Ile319, Leu320, Met323, Trp327, Leu331, Ile334 and Pro342, were mutated to Asp in order to maximally destabilize hydrophobic interactions. Consistent with expectation, mutation of multiple hydrophobic residues to Asp resulted in large decreases in affinity. The double mutant I319D/L320D reduced affinity by >25-fold (Table 2; Fig. 6A). The K d for the triple mutant I319D/L320D/M323D was immeasurable due to weak binding, but greater than 3 µM, representing a ~50-fold loss of affinity (Table 2; Fig. 6A). The same was true of two other triple hydrophobic mutants tested, M323D/L331D/I334D and W327D/L331D/I334D (Table 2; Fig. 6B). The single mutant M323D has the largest effect of any single amino acid change, with a reduction in affinity of >25-fold (Table 2; Fig. 6A). Moving closer to the center of the AFF4 ELLBow , L331D and I334D reduce affinity by ~20-and 8-fold, respectively (Table 2; Fig. 6B). This highlights the role of hydrophobic residues in AFF4 ELLBow helix α1 and immediately C-terminal to it in the central cluster as the critical anchor points and affinity determinants.
Hydrophobic residues of the central cluster make smaller contributions than those highlighted above. W327D reduces affinity 4-fold, while F345D/F347D reduces it by less than two-fold. P342D led to a similar 3-fold drop ( Table 2; Fig. 6B). These more modest contributions may reflect that these side-chains are partially solvent-accessible in the AFF4 ELLBow :ELL2 Occ complex. Moreover, their interactions are made in part with other residues within the AFF4 ELLBow such that they could potentially make residual hydrophobic interactions even in unbound AFF4. The polyproline helix does not seem to have a major role in affinity, with the double 328-329 Pro-Gly mutant reducing affinity only by a factor of three (Table 2; Fig. 6B).
The interface has a significant polar component, with some hydrophilic residues contributing substantially to binding, and others less so. The AFF4 ELLBow α1 mutant D317P/E317P was designed to disrupt hydrogen bonding involving Asp317 and to introduce helix breaker mutants in α1. This mutation lowered affinity by 10-fold (Table   2; Fig. 6A). The charge reversal mutation E322H reduced affinity by less than two-fold ( It proved impossible to purify hydrophobic to Asp mutants in the AFF4 binding site of ELL2 Occ because these proteins were insoluble when expressed in E. coli. Presumably this is because these hydrophobic residues also contribute to the hydrophobic core of the ELL2 Occ fold. It was, however, possible to purify ELL2 Occ polar mutants in the binding site. We examined the roles of ELL2 His559, His608, Asn619, and Lys625 by pull-down assay (Fig. 6C). Single mutants H559E, H608E, N619A, and K625T had no apparent effect on binding by pull-down. However, the quadruple mutant H559E/H608E/N619A/K625T completely abrogated binding in this assay. This validates the role of these residues in the interface in solution.

AFF4 ELLBow and ELL2 Occ are important for in vivo complex assembly
It had previously been shown that the AFF4 sequence 318-337 was sufficient for ELL2 binding . We tested whether this sequence was also necessary for binding, and found to our surprise that AFF4 Δ318-337 still pulled down ELL2 in nuclear extracts ( Fig. 7-S1A). We hypothesized that AFF4 contained a second ELL2 binding site, and determined that a double deletion of residues 318-337 and 970-1163 abrogated the interaction completely (Fig. 7A). In order to determine if single residues within AFF4 ELLBow contributed to binding and function in cells, point mutants were constructed in the context of AFF4 Δ970-1163. ELL1 contains a C-terminal domain homologous to that of ELL2, hence binding to ELL1 was also tested. L320D was most effective, blocking both ELL1 and ELL2, consistent with its very strong effect on binding in vitro (Fig. 6A, Table   2). E322H, P329G, and I334D partially blocked ELL2 binding but completely knocked out ELL1 binding, consistent with their intermediate effects on in vitro peptide binding.
In order to determine if the AFF4 binding site on ELL2 was functional in cells, polar mutants were inserted into ELL2 alleles and these were transfected into HeLa cells.
We avoided testing hydrophobic mutants of ELL2 since we had previously found that these destabilized the ELL2 structure. HA-tagged ELL2 H559E/H608E and ELL2 H559E/H608E/N619A/K625T were expressed at essentially wild-type levels in HeLa cells ( Fig. 7B). Wild-type HA-ELL2 pulled down AFF1, AFF4, and ELL1 from extracts. ELL2 H559E/H608E has sharply reduced binding to AFF1, AFF4, and ELL1. ELL2 H559E/H608E/N619A/K625T has only trace binding to AFF1 and AFF4 in extracts. These findings support that the structural interface is responsible for the interaction of ELL2 with both AFF1 and AFF4 in cells.

Role of the ELL2 Occ ELLBow-binding interface in HIV proviral transactivation
Overexpression of AFF4 stimulates proviral transcription by ~5-9-fold and ~26-fold in HEK 293T and HeLa cells, respectively (Fig. 8A). Unsurprisingly, AFF4 ELLBow mutants that on their own do not abrogate binding have little or no effect on transcription ( Fig. 7-S1B). Deletion of the C-terminal ELL1/2 binding domain almost completely blocked transactivation (Fig. 8A). The residual activity of AFF4Δ970-1163 was so low that meaningful results could not be obtained for transactivation phenotypes of these mutants (Fig. 8A). The abundance of the SEC complex appears to be limiting for transactivation such that overexpression of ELL2 in the presence of extra AFF4 promotes transcription by a factor of 14 (Fig. 8B). Polar mutants in the AFF4 binding site of ELL2 Occ were tested for their effects on transcription. ELL2 H559E/H608E and ELL2 H559E/H608E/N619A/K625T had 3-fold and 5-fold less transactivation activity, respectively, than wild-type. These observations strongly support a functional role for the AFF4 ELLBow binding site on ELL2 Occ in transactivation.

Discussion
The crystallization of the AFF4 ELLBow :ELL2 Occ complex rounds out our structural-level understanding of how the AFF4 scaffold recruits its three known partners in the SEC, P-TEFb, ENL/AF9, and ELL1/2. The limited solubility of ELL2 Occ made this a more challenging target for crystallization, hence the necessity for the fusion approach. When using protein chimeras as a basis for structure solution, it is particularly critical to validate the findings in solution and in functional assays. Binding assays in vitro, pulldowns from nuclear extracts, and proviral transactivation assays present a unified, consistent picture that validates the structural results.
The structure confirms the decade-old prediction that the C-terminal domains of ELL1/2 would have the same fold as the occludin ZO-1 binding domain. Occludin is a transmembrane tight junction protein that has no known involvement in transcription. It is not clear why this protein and ELL1/2 should share a domain uniquely present in this small set of otherwise unrelated proteins. In the initial analysis of the occludin structure, it was proposed that another tight junction protein, ZO-1, bound to a basic patch at the concave center of the arch (Li et al., 2005). This patch of occludin includes Lys504 and Lys511, which correspond structurally to the functionally important His618 and Lys625 in the AFF1/4 binding site of ELL2. Subsequently, another report proposed that ZO-1 bound elsewhere, at one tip of the occludin domain arch. Despite these uncertainties, the structural similarities are extensive enough to suggest a common evolutionary origin and related protein-binding functions for the three-helical domains of occludin and ELL1/2. The bromodomain and extraterminal (BET) protein inhibitor JQ1 (Filippakopoulos et al., 2010) and related compounds promote reactivation of HIV-1 from latency via P-TEFb (Banerjee et al., 2012;Bartholomeeusen et al., 2012;Boehm et al., 2013;Li et al., 2013;Zhu et al., 2012). Other small molecule activators of HIV-1 transcription are being sought in the context of HIV eradication strategies. We observed a cavity at the AFF4-ELL2 interface that appears likely to be present also in the AFF1-ELL2 complex relevant to proviral activation (Li et al., 2016), on the basis of the complete identity of the AFF1 and AFF4 residues involved. If so, this could provide an avenue for the design of new SEC activators with JQ1-like effects on latency, but acting by an orthogonal molecular mechanism.

Cloning and protein purification
DNAs for ELL2 fragments and AFF4-ELL2 fusions were subcloned into pGST-parallel2, and DNAs for AFF4 peptide fragments were subcloned into pRSFduet-1 and pHis-parallel2. Plasmids expressing FLAG-tagged wild-type AFF4 and HA-tagged wild-type ELL2 were generated previously (He et al., 2010). The plasmids expressing mutant versions of AFF4 and ELL2 were generated by PCR mutagenesis. The mutant constructs were verified by DNA sequencing. All proteins were expressed in E. coli BL21-gold coli BL21-gold (DE3) cells grown in M9 minimal medium supplemented with 5% LB medium. 0.2 mM IPTG and 100 mg selenomethionine were added when the OD 600 reached 1.0. Cells were pelleted by centrifugation at 4000 x g for 10 minutes after overnight induction at 16 °C. Se-Met AFF4 301-351 -(Gly-Ser) 4 -ELL2 519-640 was prepared as above and SeMet incorporation verified by mass spectrometry.
After 10 s, exchange was quenched at 0°C with the addition of ice-cold quench buffer (400 mMKH 2 PO 4 /H 3 PO 4 [pH 2.2]). Quenched samples were injected onto a highperformance liquid chromatography (HPLC) (Agilent 1100; Agilent Technologies) with in-line peptic digestion and desalting. Desalted peptides were eluted and directly analyzed by an Orbitrap Discovery mass spectrometer (Thermo Scientific). Peptides were identified using tandem MS/MS and Proteome Discoverer 2.1 (Thermo Scientific). Initial mass analysis of the peptide centroids was performed using HDExaminer2.0 (Sierra Analytics) followed by manual verification of every peptide. The deuteron content was adjusted for deuteron gain/loss during digestion and HPLC. Both non-deuterated and fully deuterated fusions were analyzed. Fully deuterated samples were prepared by three cycles of drying and resolubilization in D 2 O and 6 M guanidinium hydrochloride.

Crystallization of the AFF4 ELLBow -ELL2 Occ fusion
The purified fusion construct AFF4(301-351)-(Gly-Ser) 4 -ELL2(519-640) was concentrated to 10 mg/ml with a 10 kD centrifugal filter (Millipore). Crystals were grown by hanging-drop vapor-diffusion at 19°C. The protein solution was mixed with well buffer composed of 0.2 M NaCl, 10 mM MgCl 2 , 0.3M Na 3 Citrate, 0.2M Na thiocyanate, 0.1M Hepes pH 7.4. Crystals appeared in 24 hr and grew to full size in 5 days. Crystals were flash-frozen with liquid N 2 in well buffer. Se-Met crystals were grown in the same condition as native crystals.

Data collection and structure determination
Native data were collected on BL7-1 at Stanford Synchrotron Radiation Lightsource.
Native crystals diffracted to 2.5 Å and data were collected at a wavelength of 1.1271 Å.
Se-MAD data were collected on BL8.3.1 at the Advanced Light Source, LBNL, Berkeley.
The peak data set and the high energy remote data set were collected at wavelengths of 0.9797Å and 0.9569Å respectively. All data sets were processed with HKL2000 (HKL Research). Data collection and processing statistics are given in Table 1. Phases were determined by two-wavelength multiwavelength anomalous dispersion (MAD). Model building and refinement were finished with ARP/wARP (Langer et al., 2008), COOT (Emsley et al., 2010), REFMAC5 (Murshudov et al., 1997) and PHENIX (Adams et al., 2010).

Pulldown Assays
Mutants of ELL2 (519-640) and AFF4 (300-351) were purified as described above. The concentration of proteins and peptides was determined by UV absorption at 260-280-nm. 9 µM GST-ELL2 and 20 µM His 6 -AFF4 were incubated with GS4B resin at 4°C for 2 hours in 80 µL of 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5mM TCEP-HCl. The resin was washed 3 times with the incubation buffer. Then, the resin was boiled in 30 µL 1x SDS loading buffer at 95°C for 5 min before being applied to SDS-PAGE for analysis.

Fluorescence Polarization
Protein binding was measured using the fluorescence anisotropy of a 33-residue segment of AFF1 (residues 358-390) encompassing the protein-protein contacts in the crystal structure. AFF1 358-390 are almost identical to AFF4 318-350 with only 3 amino acid changes between the two homologs. The AFF1 peptide was synthesized at the University of Utah DNA/Peptide Facility using the following sequence: C-FAM-GABA-EILKEMTHSWPPPLTAIHTPSTAEPSKFPFPTK-amide where FAM indicates 5carboxyfluoroscein and GABA indicates a γ-amino-butyric acid spacer. Increasing amounts of purified Sumo-ELL2 519-640 were incubated for 30 min with 5 nM labeled peptide at room temperature in 25 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 0.05% NP40, and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP) to determine a suitable protein concentration for competition experiments. Competition titration experiments with unlabeled His-tagged AFF4 protein 301-351 were performed using 2 µM Sumo-ELL2 in 25 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 0.05% NP40, 0.5 mM TCEP, and 5 nM fluorescent peptide. Fluorescence anisotropy was measured using a Victor 3V (Perkin Elmer) multi-label plate reader. Data points represent the average of three experiments. Binding curves were fit to a formula describing competitive binding of two different ligands to a protein using Prism version 5.0c (Graphpad Software). Error bars are representative of the standard error from the mean of three experimental replicates.

Co-immunoprecipitation
Approximately 2 × 10 7 HEK 293T cells in two 145-mm dishes were transfected by plasmids expressing the wild-type or mutant FLAG-AFF4 or ELL2-HA (20 µg/each). 48 hours after transfection, the cells were harvested and swollen in 4 ml hypotonic buffer A (10 mM HEPES-KOH [pH 7.9], 1.5 mM MgCl 2 , and 10 mM KCl) for 5 minutes and then centrifuged at 362 × g for 5 min. The cells were then disrupted by grinding 20 times with a Dounce tissue homogenizer in 2 ml buffer A, followed by centrifugation at 3,220 × g   The highlighted boxes within AFF4 and ELL2 represent the co-crystallized elements described below. Other regions of AFF4 are annotated for binding to P-TEFb, AF9/ENL, and the novel C-terminal ELL1/2 binding site described below. B. Deuterium uptake data for the AFF4-ELL2 fusion complexes. HDX-MS data are shown in heat map format, where peptides are represented using rectangular strips above the protein sequence.
Absolute deuterium uptake (in %) after 10 s is indicated by a color gradient below the protein sequence. Protein sequence corresponding to the AFF4 is indicated by purple box.
Protein sequence corresponding to the N-terminal portion of the ELL2 α1 helix is indicated by red box.

Figure 6. Contributions of AFF4 ELLBow interactions to binding in solution.
A. Binding of AFF4 ELLBow WT and mutants in the N-terminal α-helix to Sumo-ELL2 Occ .