Structure of the T binding domain of Hexim1 and molecular basis for its recognition of P-TEFb

Sonja A. Dames*†, Andre´ Scho¨ nichen‡, Antje Schulte‡, Matjaz Barboric§, B. Matija Peterlin§, Stephan Grzesiek*, and Matthias Geyer†‡

*Department of Structural Biology, Biozentrum Basel, University of Basel, 4003 Basel, Switzerland; ‡Abteilung Physikalische Biochemie, Max-Planck-Institut fu¨r molekulare Physiologie, 44227 Dortmund, Germany; and §Departments of Medicine, Microbiology, and Immunology, Rosalind Russell Medical Research Center, University of California, San Francisco, CA 94143

Edited by Ann E. McDermott, Columbia University, New York, NY, and approved July 11, 2007 (received for review March 1, 2007) Hexim1 is a cellular that associates with the positive tran- is suggested to have a self inhibitory function; a central nuclear scription elongation factor b (P-TEFb) to regulate RNA polymerase II localization signal (NLS, 150–177) that interacts with the nuclear elongation of nascent mRNA transcripts. It directly binds to transport machinery and directly binds to 7SK snRNA; a region of of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest highest homology (185–220), including a negatively charged cluster of transcription elongation. Here, we report the solution structure of that might be involved in P-TEFb inhibition; and a C-terminal the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel Cyclin T binding domain (TBD) (255–359) that leads to dimeriza- coiled-coil homodimer composed of two segments and a preceding tion of Hexim molecules (7–10, 15–18). The Cyclin T binding region alpha helix that folds back onto the first coiled-coil unit. NMR constitutes a stable domain and was suggested to form a coiled-coil titration, fluorescence, and immunoprecipitation experiments re- structure. vealed the binding interface to Cyclin T1, which covers a large surface Here, we present the solution structure of the dimeric Cyclin on the first coiled-coil segment. Electrostatic interactions between an TBD of Hexim1 255–359 that consists of two coiled-coil segments acidic patch on Hexim1 and positively charged residues of Cyclin T1 and an adjacent helix. NMR mapping experiments and mutagenesis drive the complex formation that is confirmed by mutagenesis data studies in vitro and in cells revealed the binding interface to Cyclin on Hexim1 mediated transcription regulation in cells. Thus, our T1, which covers a large and highly charged surface on the first studies provide structural insights how Hexim1 recognizes the Cyclin coiled-coil segment. Hexim1 recognizes Cyclin T1 through an T1 subunit of P-TEFb, which is a key step toward the regulation of association process that is mainly driven by electrostatic interac- transcription elongation. tions. Thus, our studies provide structural insights how Hexim1 targets P-TEFb for the regulation of transcription elongation. Cyclin T1 ͉ NMR spectroscopy ͉ transcription elongation Results ranscriptional elongation by RNA polymerase II (pol II) is a The Hexim1-TBD Forms a Bipartite Coiled-Coil with a Preceding Helix. Thighly regulated process that generates full-length RNA tran- The 3D structure of the TBD of human Hexim1 (residues 255–359) scripts and coordinates transcription with pre-mRNA processing was determined by using heteronuclear NMR spectroscopy in (1). The positive transcription elongation factor b (P-TEFb) is a key solution. The structure was calculated by using 2,973 restraints and regulator for the transition from abortive to productive elongation refined in a water shell, yielding a final ensemble of 20 lowest- of mature mRNA molecules (2). The core form of P-TEFb is a energy structures with 96.3% of the backbone ⌽-/⌿-angle combi- heterodimer between the cyclin-dependent kinase 9 (Cdk9) and its nations in folded segments occupying the most favored region of the regulatory subunit Cyclin T1 (CycT1) or the minor forms T2 or K. Ramachandran plot (SI Table 1). Transcription activation by P-TEFb depends on the kinase activitiy The structure of the Hexim1-TBD is a parallel bipartite ho- of Cdk9, which phosphorylates the C-terminal domain (CTD) of modimeric coiled-coil that forms a left-handed superhelix with a the largest subunit of RNA pol II to stimulate the processivity of length of 95 Å and a diameter of Ϸ20 Å (coiled-coil parameters are elongation (3). Initially, P-TEFb has been found to play a critical given in SI Table 2). The canonical ␣-helical fold of the coiled coil role in stimulating the transcription of HIV-1 , where the viral is split into two segments of approximately similar length. Helices transactivator protein Tat recruits P-TEFb to stalled RNA pol II ␣2/␣2Ј comprise residues K284 to K313 and helices ␣3/␣3Ј residues molecules (2). At present, P-TEFb is considered a global transcrip- D319 to Q348, each forming eight turns of Ϸ40 Å length. The tional elongation factor important for the expression of most RNA coiled-coil region is preceded by a short ␣-helix (␣1) from residue pol II-regulated genes (4). T276 to N281 that docks onto the first coiled-coil segment of the Recently, it has been found that P-TEFb itself is subject to a tight mutual opposite helix ␣2Ј (Fig. 1B and SI Fig. 6). The N-terminal regulation, because approximately half of nuclear P-TEFb in HeLa cells is sequestered in an inactive state. This inactive complex is composed of the Cdk9/CycT1 heterodimer bound to the abundant Author contributions: S.A.D., B.M.P., and M.G. designed research; S.A.D., A. Scho¨nichen, A. Schulte, M.B., and M.G. performed research; S.A.D., A. Scho¨nichen, A. Schulte, M.B., and small nuclear RNA 7SK and the regulatory protein Hexim1 (5–10). M.G. analyzed data; and S.A.D., M.B., S.G., and M.G. wrote the paper. Whereas 7SK is supposed to serve as a molecular scaffold to The authors declare no conflict of interest. mediate the interaction of Hexim1 with P-TEFb, Hexim1 binds This article is a PNAS Direct Submission. directly to the cyclin box repeats of CycT1 and inhibits the kinase Freely available online through the PNAS open access option. activity of Cdk9. The other half of P-TEFb heterodimers is cata- Abbreviations: CycT1/A, Cyclin T1/A; P-TEFb, positive transcription elongation factor b; TBD, lytically active and binds the bromodomain protein, Brd4 (11, 12), Cyclin T binding domain. which has high affinity for the acetylated tails of core histones H3 Data deposition: The atomic coordinates and NMR restraints have been deposited in the and H4 and can bind the Mediator complex. , www.pdb.org (PDB ID code 2GD7). Hexim1 was initially identified as a protein whose expression is †To whom correspondence may be addressed. E-mail: [email protected] and induced in vascular smooth muscle cells in response to hexameth- [email protected]. ylene bisacetamide treatment (13, 14). Human Hexim1 consists of This article contains supporting information online at www.pnas.org/cgi/content/full/ 359 aa and is tentatively divided into four regions [supporting 0701848104/DC1. information (SI) Fig. 5A]: a variable N-terminal region (1–149) that © 2007 by The National Academy of Sciences of the USA

14312–14317 ͉ PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701848104 Downloaded by guest on September 24, 2021 (Y291), and both are highly evolutionary conserved (SI Fig. 5B). The observed sequence of residues at the a position (K284, Y291, L298, N305) might therefore confer dimerization specificity and define the start of the coiled-coil at the expense of a lower stability because of less tight ␣-helical packing. The d positions in this segment are occupied by leucines and methione, which are both favored at this position. Most of the interface of the second coiled-coil segment (␣3/␣3Ј) shows good conformity with the heptad consensus sequence. The first three repeats contain leucines at the d position and valine, leucine, and asparagine at the a position. The last heptad repeat d position is occupied by histidine, which is commonly not found at this position and might thereby promote the termination of the dimeric coiled-coil. The e and g positions are typically occupied with charged residues and influence the pairing specificity. Attractive ionic interactions between heptad repeat position g of one strand and eЈ of the opposite one (denoted as i 3 iЈϩ5) stabilize the coiled-coil dimer, whereas repelling ones destabilize it. Although E290 and E295 in the N-terminal coiled-coil form a repulsive pair, only E304 and R309 strengthen the binding interface (Fig. 1D). The situation is more uniform on the second coiled-coil segment where three favorable ionic interactions are formed (R321–E326, E328–R333, E342–R347).

Backbone Dynamics and Structural Integrity. The flexibility of the TBD backbone was assayed by the analysis of 15N relaxation data 1 15 (SI Fig. 7A).AfitofT2 and T1 relaxation times and [ H] N NOEs BIOCHEMISTRY Fig. 1. NMR structure of the TBD of human Hexim1. (A) Ribbon represen- by the program TENSOR2 (21) showed that rotational diffusion of tation of the dimeric TBD (255–359). Secondary structure elements of both the TBD is significantly better described by an axially symmetric 2 2 chains are labeled red and blue, respectively. The stammer (316–318) between diffusion tensor than with an isotropic model [␹ (ani)/ ␹ (iso) ϭ the two coiled-coil segments ␣2 and ␣3 leads to an overwinding of the 0.3]. A fully anisotropic model did not further improve the fit. The supercoil. (B)(Left) Backbone atoms of the 20 lowest energy structures were long axis of the axially symmetric diffusion tensor is parallel to the superimposed for the first coiled-coil segment (276–313; rmsd, 0.38 Å; see also long axis of the molecule with rotational correlation times ␶ʈ ϭ 7.2 SI Table 1). (Right) Superposition of those for the second (319–348; rmsd, 0.32 ns and t֊ ϭ 21.4ns(SI Table 3). These calculations are in good Å), including a rotation by 90° compared with Left. Side chains of residues at agreement with values expected for the size and shape of the TBD the coiled-coil heptad repeat positions a and d are depicted in dark and light dimer. Large amplitude backbone motions on the subnanosecond green, respectively. (C) Electrostatic surface charge of the TBD dimer (255– time scale are evident from decreased [1H]15N NOEs and increased 359). Note the large negatively charged surface patch (red) in the first coiled- 15 coil segment that is confined by positive charges (blue). (D) Helical wheel NT2 for the N-terminal 20 and the C-terminal 10 residues and the representation of the coiled-coil segments from 284–311 (␣2/␣2Ј)(Left) and stammer (316–318) in between the two coiled-coil segments. 1 15 from 319–348 (␣3/␣3Ј)(Right). Positions a to g of residues within the heptad A superposition of the H/ N HSQC spectra of fragments repeat pattern are indicated. Salt bridges between g and eЈ positions are 242–359, 255–359, and 255–316 confirmed the presence of the depicted by dashed lines. coiled-coil structure and the preceding ␣1-helix in all three frag- ments (SI Fig. 7B), including the construct lacking the second coiled-coil segment. The N-terminally extended fragment 242–359 20 and the C-terminal 10 residues appeared flexible and not did not exhibit additional folded conformation, because all addi- restrained by intermediate or long range NOEs, but truncation or tional 13 residues showed chemical shift values in the random coil extension of the N-terminal linker segment severely influenced the range (Ϸ8.20–8.52 ppm for 1H). expression yield and solubility properties of this domain. Mapping of the Cyclin T1 Binding Interface by NMR Titration Experi- The TBD Coiled-Coil Dimer Interface. Left-handed coiled-coil struc- ments. Upon CycT1 binding, resonances of the free Hexim1-TBD tures are characterized by a repeating pattern of seven residues, weakened continuously, indicating intermediate exchange with the denoted (abcdefg)n. Residues at positions a and d are typically bound form on the NMR timescale (SI Fig. 8). New resonances that nonpolar and form the oligomerization interface of the helices, would represent the TBD in the CycT1–TBD complex did not whereas residues at e and g are largely solvent exposed, polar appear, most likely because of a low stability of CycT1 and residues that guide the oligomerization specificity through electro- unspecific aggregation of the 60-kDa protein complex, which would static interactions (19). Parallel coiled-coil structures display a broaden the signals beyond detection. Other factors that could have distinctive ‘‘knobs-into-holes’’ packing, in which the side chains of influenced the observed loss in signal intensity are local motions residues at the heptad repeat a and d positions form successive and the degree of separation between the chemical shifts of the free layers. Although residues 284–348 of Hexim1 adopt the structure and the bound form. For a quantitative evaluation, the ratio of the of a typical coiled-coil domain (Fig. 1 A–C), the sequence of the TBD resonance peak intensities [I(TBDfree)/I(TBDbound)] was cal- interacting dimer surface varies from the strict consensus (Fig. 1D). culated at a molar ratio of 0.5 CycT1 to TBD (Fig. 2A). Because of In the first coiled-coil segment (␣2/␣2Ј), one a position is occupied the overlap of resonances, some peak intensities could not be by leucine, and one by asparagine. Both residues are often observed determined and were omitted in the diagram. The largest intensity at this position in dimeric coiled-coils (20). However, none of the changes were observed for residues within the first two heptad four amino acids occupying an a position is ␤-branched (Ile, Val, repeats (KEYL, 289–292) of the first coiled-coil segment. In Thr) as favored for a tight packing in a coiled-coil dimer (19). addition, large intensity changes were observed for the backbone Instead, one a position is occupied by a charged residue (K284), and amides of residues E295, K296, S299, E302, and N306. All these the following one is occupied by a bulky polar aromatic residue residues are located on the first coiled-coil segment of the TBD,

Dames et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14313 Downloaded by guest on September 24, 2021 Fig. 3. Mutational analysis of the Hexim1-TBD/Cyclin T1 interaction. (A) Stopped flow dissociation competition experiments among CycT1, fluorescent- labeled TBD, and excess mutant TBDs. The preformed equimolar complex of CycT1 and TBD-EDANS was mixed with a 20-fold excess of nonlabeled TBD or buffer solution. The dissociation course was monitored for 0.5 s. The dilution and Fig. 2. The binding interface of Cylin T1 on the Hexim1-TBD. (A) Loss of the competitive dissociation of TBD-EDANS from CycT1 by nonlabeled TBD mol- 1 15 H/ N HSQC peak intensity upon titration of unlabeled CycT1 (1–292) to ecules led to a characteristic decrease of the fluorescence signal. Binding con- 15 N-labeled TBD (255–359) at a molar ratio of 0.5. The stronger the decrease stants and kinetic parameters are listed in SI Table 4.(B) Binding kinetics between of the peak intensity compared with the spectrum without CycT1, the higher TBD and wild-type CycT1 (1–292) or mutant CycT1(KR4A) (Left) as recorded by the plotted intensity ratio. (B) Display of the CycT1 binding surface on the stopped flow measurements. The binding affinity to CycT1(KR4A) is Ϸ20-fold Hexim1-TBD structure as identified by NMR perturbation measurements. The reduced compared with the wild-type protein, mainly because of a significantly color coding corresponds to an intensity ratio for low (intensity ratio 4–10, decreased association rate kon (Right). yellow), medium (Ͼ10–15, orange), and large changes (Ͼ15, red). (C) Surface and ribbon representation of the sequence conservation of Hexim-TBDs. Sequence identities based on the evaluation of 18 Hexim genes are colored surface of the TBD structure and on sequence conservation, 16 according to the degree of conservation for pink (variable, 17%), gray (aver- individual mutants of the TBD ranging from positions E277 to age, 66%), and blue (conserved, 100%). N311 were constructed. For direct comparisons and to minimize the influence on the structural integrity of the TBD, only alanine suggesting that this structure forms the recognition site for CycT1. mutations were generated. Mutations at core forming positions that Two other resonances that showed increased intensity changes are would possibly destabilize the coiled-coil dimer were omitted. In L279 and S283, located on the preceding helix ␣1 and the connect- addition, a triple mutant E286A/E290A/E293A (named 3E-A) that ing loop to the coiled-coil section. destroys the negatively charged surface patch on the first coiled-coil The results of the NMR titration were mapped on the surface of segment was generated. First, GST-fused CycT1 coupled to GSH- the TBD structure (Fig. 2B). Of the 12 most affected residues, 3 are Sepharose beads was used to pull down wild-type or mutant TBD located at heptad repeat b positions (L292, S299, N306), and 4 protein that had been added in equal amounts (SI Fig. 9). additional residues (E295, E302, K289, K296) are on the adjacent For a quantitative analysis of the mutant binding a fluorescence e and f positions. Y291 was the only amino acid at a core forming competition assay (22) was developed that is based on the displace- a position that was perturbed upon binding, inline with the sug- ment of wild-type TBD from its binding site on CycT1 by the gestion that large aromatic and hydrophilic amino acids at these addition of excess mutant TBD (Fig. 3A). Dissociation with wild- positions destabilize the coiled-coil interface. Mapping the identity type TBD or buffer solution decreased the fluorescence signal conservation onto the structure of the TBD revealed that residues intensity by 23% and 4%, respectively. Several of the TBD mutant on the surface of the first coiled-coil segment are more conserved showed a significantly diminished ability to displace TBD- than those of the second, whereas residues in the coiled-coil EDANS from CycT1. For eight of the mutant proteins, the disso- interface at a and d positions are overall well conserved (Fig. 2C and ciation constant Kd was Ͼ4-fold higher than for wild-type TBD (SI SI Text). A continuous surface of high sequence conservation that Table 4). Knowing the association and dissociation rates of wild- is formed by residues on helices ␣1Ј and ␣2 overlaps well with type TBD to CycT1 from previous (15) and present experiments, residues that were found to interact with CycT1 based on the NMR the kinetic parameter for the binding of mutant Hexim1 could be titration data. calculated from the slope of the curve. L292A showed a 4-fold higher koff rate but a similar kon rate, which is typical for the loss of Mutational Analysis of the Binding Interface Between Hexim1-TBD and a hydrophobic interaction upon mutation. K289A, K296A, and Cyclin T1. The binding interface between TBD and CycT1 was 3E-A all showed reduced kon rates indicating the efficacy of further characterized by mutagenesis studies. Based on the NMR electrostatic interactions for the association of the two molecules. titration data, the proximity of CycT1 binding residues on the The loss of electrostatic attraction upon mutation of a negatively

14314 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701848104 Dames et al. Downloaded by guest on September 24, 2021 BIOCHEMISTRY

Fig. 4. Functional analysis of mutant Hexim1 proteins in cells. (A and B) Multiple amino acid substitutions in the Hexim1-TBD decrease the ability of Hexim1 to bind P-TEFb in cells. f.Hxm1 proteins that were expressed in HeLa cells from corresponding plasmid effectors (6 ␮g) and immunoprecipitated by anti-FLAG M2 beads are indicated above the Western blots. Arrows to the left indicate the bound P-TEFb and the amounts of immunoprecipitated f.Hxm1 proteins, respectively. (C–E) Multiple amino acid substitutions in the TBD disable the inhibitory function of Hexim1 in cells. HeLa cells expressed plasmid reporter pG6TAR (0.2 ␮g). Proteins that were coexpressed from corresponding plasmid effectors (Gal4.CycT1, 0.5 ␮g; f.Hxm1, 0.8 ␮g) with the plasmid reporter are presented below the CAT data. (Lower) Shown is the expression of f.Hxm1 proteins as indicated by the arrow.

charged cluster in the binding interface of the TBD prompted us to (Fig. 1C). Moreover, we mutated L292 and N306 within the second test for a positively charged region in the cyclin box repeats of and fourth heptad repeat, respectively. The selected residues were CycT1. Residues K247, R251, K253, and R254 of CycT1 (1–281) substituted by alanines individually or in combination. were mutated to alanines, termed KR4A, and the binding kinetics Mutated and wild-type f.Hxm1 proteins were expressed in HeLa for the interaction with the TBD were determined by stopped flow cells, immunoprecipitated from total cell lysates, and tested for binding to endogenous P-TEFb by Western blot analysis with measurements (Fig. 3B). The association rate kon was reduced Ͼ6-fold and the dissociation rate k increased Ϸ3-fold, resulting in antibodies directed against CycT1. As expected, wild-type f.Hxm1 off bound CycT1 in cells (Fig. 4A, lane 2). None of the tested single an apparent Kd of 68 ␮M for the mutant protein. These results strongly support the electrostatic nature in the association of CycT1 amino acid substitutions abrogated binding completely (Fig. 4A, lanes 3–7), but mutation of the acidic residues E295A or E302A and TBD and help to explain previous observations of mutually decreased the binding affinity (lanes 5–6). The combined mutation exclusive binding of either Hexim1-TBD or HIV-1 Tat to CycT1 of E290 and E295 reduced the binding further (Fig. 4B, lane 4), (15) by mapping the interaction to a similar region. whereas the double mutation E302A/N306A did not show a sig- nificant effect (lane 6). When three or four residues were substi- Binding of Mutant Hexim1 to P-TEFb in Cells. The importance of the tuted together, binding to CycT1 was in general strongly reduced identified residues of Hexim1 to bind, and thus inhibit P-TEFb was (Fig. 4B, lanes 7–10). Levels of f.Hxm1 proteins in the immuno- further investigated by expressing selected full-length FLAG precipitations were comparable (Fig. 4 A and B Lower). epitope-tagged Hexim1 (f.Hxm1) mutants in HeLa cells. Because the interaction between TBD and CycT1 is most likely of electro- P-TEFb Inhibition Requires Residues in the N-Terminal TBD Coiled-Coil static nature, we mutated residues E290, E295, and E302, which Segment. The contribution of the identified residues on the inhib- form part of a negatively charged patch on the surface of the TBD itory property of Hexim1 was addressed by transcriptional assays in

Dames et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14315 Downloaded by guest on September 24, 2021 HeLa cells, using a system consisting of chimeric Gal4.CycT1 to inhibit P-TEFb in the presence of 7SK snRNA (16). Because protein and the plasmid reporter pG6TAR, which contains six Gal4 these mutations are at the dimerization interface, the structural DNA binding sites positioned upstream of the HIV-1 long terminal integrity of the first TBD coiled-coil segment seems to be required repeat (HIV LTR), followed by the chloramphenicol acetyltrans- for P-TEFb recognition. A cooperative interaction between ferase (CAT) reporter . The recruitment of Gal4.CycT1 to the Hexim1 and P-TEFb for two individual binding sites at the 5Ј and pG6TAR promoter activates transcription that depends on the 3Ј hairpin loops of 7SK snRNA was suggested recently (24). 7SK kinase activity of P-TEFb (23). When we expressed the Gal4.CycT1 snRNA might thus act as a stabilizing factor that gains additional chimera together with pG6TAR in HeLa cells, the levels of CAT specificity for the CycT1–Hexim1 interaction. activity increased 32-fold over the basal levels, whereas coexpres- But how might Hexim1, bound to the CycT1 subunit of P-TEFb, sion of the wild-type f.Hxm1 protein decreased this activity to 4-fold repress the Cdk9 kinase activity? The presence of the TBD within (Fig. 4C, bars 1 and 2). Alanine substitutions of E290, L292, E295, Hexim1 was found to be required for inhibition of P-TEFb in cells and E302 reduced the ability of f.Hxm1 to inhibit P-TEFb in cells, (15) and an N-terminal truncation mutant of Hexim1 (181–359) whereas the substitution of N306 had no effect (Fig. 4C, bars 3–7). inhibited P-TEFb in vitro independently of 7SK snRNA (10, 16). In When compared with the inhibition by the wild-type f.Hxm1, this analogy to the inhibition of the Cdk2/CycA complex by p27Cip2 (25), effect ranged from 1.6-fold (L292) to 5-fold (E295). The mutations it has been suggested that tyrosines N-terminal of the determined K289A or K296A only slightly affected transcription inhibition, but CycT1 binding site (Y203, Y271) might block the ATP binding site effects were more pronounced by using lower amounts of trans- in Cdk9 (10, 16). As shown here, the TBD of Hexim1 binds in close fected plasmids (SI Fig. 10). Based on circular dichroism data the vicinity to the TRM region of the C-terminal helices of the CycT1 mutant K289A has about the same amount of ␣-helical secondary cyclin box repeats. Therefore, a catalytically repressive motif in structure as wild-type TBD indicating that the mutation did not Hexim1 is expected to be rather distant from the CycT1 binding site disrupt the coiled-coil fold (SI Fig. 11). to cover the distance to the nucleotide binding site of the kinase that Mutant f.Hxm1 proteins with double substitutions mostly lost is supposed to reside on the opposite side of the cyclin molecule. their inhibitory abilities (Fig. 4D, bars 3–7). In agreement with the p27Cip2 inhibits Cdk2 by binding and subsequent folding of an binding studies, the biggest effect was seen when E290 and E295 elongated region of Ϸ70 residues that covers a combined Cdk2/ were mutated together, which resulted in the complete inactivation CycA surface (25). Moreover, studies with the Cdk2/CycA substrate of f.Hxm1 (lane 4). The combination of double substitutions (Fig. CDC6 showed that recognition and discrimination against different 4E) or the triple mutant E286/E290/E293 (SI Fig. 10) rendered substrates are achieved by a recruitment site on the cyclin and a f.Hxm1 proteins nonfunctional. In summary, CycT1 recognizes a second interaction site on the kinase that are connected by a linker large surface composed of mostly acidic residues that are scattered of defined length and composition (26). For Hexim1, bridging along the first coiled-coil segment of the Hexim1-TBD structure. between a Cdk9-inactivating tyrosine and the CycT1 interface would be consistent with our observation that the region N termi- Discussion nus of the first TBD ␣-helix (242–276) that is rich in conserved Our studies showed that the C-terminal region of Hexim1 forms a residues is unstructured and flexible. Alternatively, Hexim1 could bipartite dimeric coiled-coil of which the first segment is required bind the Cdk9/CycT1 heterodimer and alter the shape of the Cdk9 for the interaction with the CycT1 subunit of P-TEFb. The residue catalytic cleft by allosteric interactions, thus interfering with kinase composition of this coiled-coil segment diverges significantly from ATP binding. Finally, binding of the Hexim1 dimer might displace that of an ideal parallel coiled-coil, exhibiting evolutionary con- the C-terminal helix of the cyclin boxes, which is known to deter- served charged and bulky polar residues at the heptad a positions mine the function and specificity of the respective Cyclin. Future (K284, Y291) and hydrophobic residues at e and b positions (I288, studies will be directed to analyze the structure of the ternary L292). Moreover, conserved charged residues at positions g and e complex formation between Cdk9, CycT1, and Hexim1 to further contribute to a negative surface patch (E290, E295) instead of enlighten the mechanism of P-TEFb inhibition. forming a stabilizing salt bridge between the dimer chains. The second segment matches almost perfectly the preferred canonical Materials and Methods consensus, suggesting a stable association of both strands. Thus, Plasmid Cloning, Protein Expression, and Purification. Plasmids en- several features that distinguish the two coiled-coil segments pro- coding Hexim1-TBD fragments (242–359, 255–359, 255–316) were vide a foundation for their functional differences. generated as described in ref. 15. Site-directed mutagenesis was In vitro binding assays and functional in vivo experiments delin- performed by using the mega-primer method similarly as described eated a large and acidic CycT1 binding interface on the first in ref. 27. The DNA coding for CycT1 (1–272 or 1–292) was cloned coiled-coil segment of the TBD. The observed small differences in into pGEX-4T1-tev (Amersham, Piscataway, NJ) and expressed as the contribution of individual residues to the binding affinity and GST-fusion protein. The plasmid reporter pG6TAR and the plas- the inhibition of transcription activity can be accounted for by the mids coding for Gal4.CycT1 and f.Hxm1 are described in ref. 18. To different experimental set-up for the in vitro and in vivo experi- construct mutant f.Hxm1 proteins, the pFLAG-CMV-2.Hxm1 plas- ments. In GST-pull down and fluorescence competition displace- mids were subjected to site-directed mutagenesis, using the ment experiments, individually purified domains were used, QuikChange II XL site-directed mutagenesis kit (Stratagene, La whereas the immunoprecipitation and in vivo transactivation anal- Jolla, CA). ysis were performed with full-length proteins in cells that harbor the Hexim1 and CycT1 proteins were expressed from Escherichia coli cofactors Cdk9 and 7SK snRNA. Previous studies showed that the BL21(DE3) cells (Novagen, Madison, WI) and purified as de- cyclin box repeat region of CycT1 (1–292) is sufficient for binding scribed in ref. 15. Uniformly 15N- or 13C/15N-labeled TBD was 15 to Hexim1 and that residues outside this domain do not contribute prepared in minimal medium containing NH4Cl as the sole 13 to the interaction with Hexim1 (9). nitrogen source or C6 D-glucose as the sole carbon source, Despite conserved differences from the heptad repeat consensus respectively. Fractions were analyzed by SDS/PAGE, and fractions sequence, the structure of the first coiled-coil segment and the containing Hexim1-TBD proteins (Ϸ98% pure) were concentrated preceding ␣-helix is preserved in a TBD construct that lacks the (Amicon filter) up to 1.0 mM in 20 mM KPi (pH 7.4) buffer/50 mM second coiled-coil segment (Hexim1 255–316, SI Fig. 7B). A NaCl/1 mM DTE and stored at Ϫ80°C. Fluorescence labeling of the functional consequence of this is that C-terminally truncated TBD TBD with the dye 1,5-IAEDANS (Molecular Probes, Eugene, OR) still binds CycT1 and inhibits transcription activity of P-TEFb (9, was performed as described in ref. 15. Protein concentrations were 15). In contrast, full length Hexim1 with two mutated heptad repeat determined by Bradford assay (Bio-Rad, Hercules, CA) and ex- d positions (L287R and L294R), showed a markedly reduced ability tinction coefficient measurements.

14316 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0701848104 Dames et al. Downloaded by guest on September 24, 2021 NMR Spectroscopy. NMR samples contained Ϸ0.6–1.2 mM restrained to one of the staggered conformations (60°, 180°, Ϫ60°) Hexim1-TBD in 20 mM KPi (pH 7.2) buffer (95% H2O/5% D2O Ϯ30°. Residual dipolar couplings were included, using the ISAC or 100% D2O) with 50 mM NaCl/10 mM TCEP⅐HCl/1 mM DTE. algorithm (37) by calling the TENSO statement. The alignment The sample for measuring residual dipolar couplings was supple- tensors for residues 277–315 and 319–348 were fitted independently mented with Ϸ16 mg/ml PF1 phages (ASLA Biotech, Riga, Latvia). during the calculation. The final values for the force constants Mapping of the binding interface was performed on 0.1 mM during the molecular dynamics in torsion angle (cartesian coordi- Ϫ Ϫ samples of 15N-labeled TBD titrated with 0–0.1 mM unlabeled nate) space were 150 (50) kcal mol 1Å 2 for NOE distance Ϫ Ϫ CycT1 (1–292). NMR spectra were acquired at 308 K on Bruker restraints, 50 (300) kcal mol 1 rad 2 for dihedral angle restraints, Ϫ Ϫ (Newark, DE) DRX600 and DRX800 spectrometers. Data were and 0.6 (1.2) kcal mol 1 Hz 2 for residual dipolar coupling re- processed with NMRPipe (28) and analyzed by using NMRView straints. The 20 best of 200 calculated structures were finally refined (29). The assignments for 13C, 15N, and 1H nuclei are described in in a water shell (38, 39). ref. 30. Distance restraints were obtained from 15N- and 13C-edited In Vitro Binding Studies and Fluorescence Measurements. Fluores- NOESY spectra. Interchain distance restraints were derived from cence experiments were performed at 20°C in 20 mM Hepes (pH a3D13C F1-edited, F3-filtered HMQC-NOESY spectrum (31, 32) 7.5) buffer containing 50 mM NaCl, 1 mM TCEP, and 1% (vol/vol) recorded on a sample containing 13C/12C mixed Hexim1-TBD glycerol. Kinetic dissociation measurements were done by using a dimers. 15N-1H residual dipolar couplings were obtained from the SX.18MV stopped flow apparatus (Applied Photophysics, Leath- 15 1 ␣ erhead, Surrey, U.K.) by rapid mixing of a preequilibrated solution analysis of N- H IPAP-HSQC (33) and 3D H -coupled HN- ␮ ␮ (CO)CA data (34) acquired on the sample containing PF1 phages. of 5 M CycT1 (1–292) and 5 M dimeric TBD-EDANS with 100 1 ␮M excess of wild-type or mutant dimeric TBD. The dansyl-based The maximal DN-H was 16 Hz. Information about the backbone dynamics was derived from the measurement of 15N-relaxation fluorophore was excited at 337 nm, and the fluorescence emission experiments, including T , T , and [1H]15N NOE (35). was recorded through a 420-nm cutoff filter. The competition 1 2 displacement curve was fitted to the fluorescence traces according to pseudo second-order kinetics. Dissociation experiments were Structure Calculations. Structure calculations were performed with analyzed with Scientist software, Version 1.0 (OriginLab, X-PLOR-NIH (36), using molecular dynamics in torsion angle and Northampton, MA). cartesian coordinate space. Distance restraints were generated by using NMRView and classified according to NOE-crosspeak in-

Transient Transfection and CAT Reporter Gene Assay. HeLa cells were BIOCHEMISTRY tensities. Upper bounds were 2.8, 3.5, 4.5, and 5.5 Å. The lower seeded into six-well plates or 100-mm-diameter Petri dishes Ϸ12 h bound was always 1.8 Å. For all NOE restraints, rϪ6 sum averaging 15 before transfection and transfected with FuGENE6 reagent (Roche was used. Initially, all distance restraints from the 3D N-edited Applied Science, Indianapolis, IN). CAT enzymatic assays were NOESY spectrum were assigned as intrachain, because backbone ␣ performed as described in ref. 23. Fold transactivation represents amides in -helices only rarely mediate interchain contacts. For the the ratio between the Gal4.CycT1-activated transcription and the next calculation step, interchain distance restraints from the 3D 13 activity of the reporter plasmid alone. Error bars give standard F1-edited, F3-filtered C-NOESY spectrum of the heterolabeled errors of the mean. dimer sample were added. Distance restraints from the 13C-edited 13 NOESY spectrum of the uniformly C-labeled sample were as- We thank Diana Ludwig for expert technical assistance; Roger Goody for signed as intra- or intermonomer or ambiguous, based on the stimulating discussions, valuable help on kinetic data evaluation, and presence of the same correlations in the 3D F1-edited F3-filtered continuous support; Q. Zhou, J.H. Yik (both at University of California, 13C-NOESY spectrum and the initial structure calculations. Back- Berkeley, CA), and H. Tanaka (University at Tokyo, Tokyo, Japan) for bone dihedral angle restraints for ␾ and ␺ were derived based on sharing plasmid reagents; and Chris A. E. M. Spronk (Center for Molecular 3 and Biomolecular Informatics, University of Nijmegen, Nijmegen, The JHNH␣, using error bounds of Ϯ25° (Ϯ30° at the helix termini) and Ϯ40°, respectively. Netherlands) for help by the structural refinement in a water shell. This work was supported Deutsche Forschungsgemeinschaft Grant GE-976/2 (to For regions with ␣-helical secondary structure, hydrogen bonds O M.G.), Schweizer Nationalfonds Grant 31-109712 (to S.G.), National In- were defined by HN-O distance bounds of 1.8–2.3 Å and N O stitutes of Health Grant R01 AI49104 (to B.M.P.), a fellowship from the 3 3 distance bounds of 2.6–3.1 Å. Based on JH␣H␤2/3 and JNH␤2/3 Treubel Fonds (Basel, Switzerland) (to S.A.D.), American Foundation for coupling constants and NOE data, side chain ␹1 angles were AIDS Research Grant 106584-36-RFNT (to M.B.).

1. Sims 3rd RJ, Belotserkovskaya R, Reinberg D (2004) Genes Dev 18:2437–2468. 20. Woolfson DN (2005) Adv Protein Chem 70:79–112. 2. Peterlin BM, Price DH (2006) Mol Cell 23:297–305. 21. Dosset P, Hus JC, Blackledge M, Marion D (2000) J Biomol NMR 16:23–28. 3. Goodrich JA, Kugel JF (2006) Nat Rev Mol Cell Biol 7:612–616. 22. Goody RS (2003) In Kinetic Analysis of Macromolecules. A Practical Approach, ed Johnson 4. Saunders A, Core LJ, Lis JT (2006) Nat Rev Mol Cell Biol 7:557–567. KA (Oxford Univ Press, Oxford), pp 153–170. 5. Yang Z, Zhu Q, Luo K, Zhou Q (2001) Nature 414:317–322. 23. Taube R, Lin X, Irwin D, Fujinaga K, Peterlin BM (2002) Mol Cell Biol 22:321–331. 6. Nguyen VT, Kiss T, Michels AA, Bensaude O (2001) Nature 414:322–325. 24. Egloff S, Van Herreweghe E, Kiss T (2006) Mol Cell Biol 26:630–642. 7. Yik JH, Chen R, Nishimura R, Jennings JL, Link AJ, Zhou Q (2003) Mol Cell 12:971–982. 25. Pavletich NP (1999) J Mol Biol 287:821–828. 8. Yik JH, Chen R, Pezda AC, Samford CS, Zhou Q (2004) Mol Cell Biol 24:5094–5105. 26. Cheng KY, Noble ME, Skamnaki V, Brown NR, Lowe ED, Kontogiannis L, Shen K, Cole 9. Michels AA, Nguyen VT, Fraldi A, Labas V, Edwards M, Bonnet F, Lania L, Bensaude O PA, Siligardi G, Johnson LN (2006) J Biol Chem 281:23167–23179. (2003) Mol Cell Biol 23:4859–4869. 27. Breuer S, Gerlach H, Kolaric B, Urbanke C, Opitz N, Geyer M (2006) Biochemistry 10. Michels AA, Fraldi A, Li Q, Adamson TE, Bonnet F, Nguyen VT, Sedore SC, Price JP, Price 45:2339–2349. DH, Lania L, Bensaude O (2004) EMBO J 23:2608–2619. 28. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) J Biomol NMR 6:277–293. 11. Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K (2005) Mol Cell 19:523–534. 29. Johnson BR, Blevins RA (1994) J Biomol NMR 4:603–614. 12. Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, Zhou Q (2005) Mol Cell 19:535–545. 30. Dames SA, Scho¨nichen A, Grzesiek S, Geyer M (2006) J Biomol NMR 36:39. 13. Kusuhara M, Nagasaki K, Kimura K, Maass N, Manabe T, Ishikawa S, Aikawa M, Miyazaki 31. Lee W, Revington MJ, Arrowsmith C, Kay LE (1994) FEBS Lett 350:87–90. K, Yamaguchi K (1999) Biomed Res 20:273–279. 32. Dames SA, Kammerer RA, Wiltscheck R, Engel J, Alexandrescu AT (1998) Nat Struct Biol 14. Ouchida R, Kusuhara M, Shimizu N, Hisada T, Makino Y, Morimoto C, Handa H, Ohsuzu 5:687–691. F, Tanaka H (2003) Genes Cells 8:95–107. 33. Ottiger M, Delaglio F, Bax A (1998) J Magn Reson 131:373–378. 15. Schulte A, Czudnochowski N, Barboric M, Schonichen A, Blazek D, Peterlin BM, Geyer M 34. Bax A, Kontaxis G, Tjandra N (2001) Methods Enzymol 339:127–174. (2005) J Biol Chem 280:24968–24977. 35. Grzesiek S, Bax A, Hu JS, Kaufman J, Palmer I, Stahl SJ, Tjandra N, Wingfield PT (1997) 16. Li Q, Price JP, Byers SA, Cheng D, Peng J, Price DH (2005) J Biol Chem 280:28819–28826. Protein Sci 6:1248–1263. 17. Dulac C, Michels AA, Fraldi A, Bonnet F, Nguyen VT, Napolitano G, Lania L, Bensaude 36. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) J Magn Reson 160:65–73. O (2005) J Biol Chem 280:30619–30629. 37. Sass H-J, Musco G, Stahl SJ, Wingfield PT, Grzesiek S (2001) J Biomol NMR 21:275–280. 18. Barboric M, Kohoutek J, Price JP, Blazek D, Price DH, Peterlin BM (2005) EMBO J 38. Linge JP, Williams MA, Spronk CAEM, Bonvin AMJJ, Nilges M (2003) Proteins 50:496–506. 24:4291–4303. 39. Nabuurs SB, Nederveen AJ, Vranken W, Doreleijers JF, Bonvin AMJJ, Vuister GW, Vriend 19. Lupas AN, Gruber M (2005) Adv Protein Chem 70:37–78. G, Spronk CAEM (2004) Proteins 55:483–486.

Dames et al. PNAS ͉ September 4, 2007 ͉ vol. 104 ͉ no. 36 ͉ 14317 Downloaded by guest on September 24, 2021