ARTICLES

Crystal structure of HIV-1 Tat complexedwith human P-TEFbTahir H. Tahirov1, Nigar D. Babayeva1, Katayoun Varzavand2, Jeffrey J. Cooper2, Stanley C. Sedore2 & David H. Price2

Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controllingtranscription elongation. The viral protein Tat hijacks the host cell’s RNA polymerase II elongation control machinerythrough interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productiveelongation of HIV mRNA. Here we describe the crystal structure of the TatNP-TEFb complex containing HIV-1 Tat, humanCdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to thesurface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of theCdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites.Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design ofcompounds that would specifically inhibit the TatNP-TEFb complex and block HIV replication.

According to the most recent UNAIDS AIDS Epidemic Update, as ofthe end of 2008, over 33 million people were living with HIV.Although the number of people infected has increased about 30%over the last 10 years, part of the increase is due to improved anti-retroviral therapies and an increase in the number of people treated.Unfortunately, strains of HIV that are resistant to current anti-HIVdrugs arise rapidly due to the error-prone reverse transcription stepin the viral life cycle, and to a very high frequency of recombinationthat leads to a generation of mutant viruses that are unresponsive tothe drugs1. Because of this and the lack of effective vaccines2, thesearch for new and better anti-HIV therapies continues. Most currentHIV drugs inhibit viral enzymes, but host proteins that interact withviral proteins are also potential targets. One such host protein is thepositive transcription elongation factor, P-TEFb, which interactswith the viral transactivator of transcription, Tat3–5.

P-TEFb has a key role in regulating the elongation phase of tran-scription by RNA polymerase II6. It is a cyclin-dependent kinasecomprised of Cdk9 paired with one of several cyclin subunits, andin its active form is responsible for relief of the block to promoterproximally paused polymerases7–9. P-TEFb is generally required forthe generation of mRNAs10 and is recruited to genes by a variety ofgene-specific proteins including NFkB, CIITA and nuclear recep-tors, as well as more generally by the bromodomain containingprotein BRD4 (refs 6, 11). The activity of P-TEFb is globally regulatedby reversible association with the 7SK snRNP (small nuclear ribo-nucleoprotein particle) in which it is held in an inhibited state by anRNA-binding protein HEXIM1 or HEXIM2 (refs 6, 11, 12).

HIV has evolved to manipulate both the Cdk9Ncyclin T1 form ofP-TEFb and the cellular process controlling P-TEFb. Expression of HIVTat in otherwise uninfected cells leads to the release of P-TEFb from the7SK snRNP and the formation of a TatNP-TEFb complex13. During anactive HIV infection, the TatNP-TEFb complex is recruited to the trans-activation response element, TAR, which is manifested in the first 59nucleotides of the nascent HIV transcript5,14–16. In addition to provid-ing a means to recruit P-TEFb to promoter proximally stalled poly-merases engaged in the HIV long terminal repeat (LTR), Tat stimulatesthe kinase activity and changes the substrate specificity of P-TEFb17.

Here we report the high-resolution crystal structures of the TatNP-TEFb complex and the TatNP-TEFb complex with bound ATP ana-logue adenylyl imidodiphosphate (AMP-PNP) and magnesium ion(further referred to as TatNP-TEFbNATP). The structures reveal thatTat interacts with both the cyclin T1 and Cdk9 subunits of P-TEFb,and provide evidence for a Tat-induced conformational change inP-TEFb. Our findings also provide a structural basis for the design ofcompounds that would specifically inhibit HIV transcription.

Crystal structure of TatNP-TEFb complex

The structure of the TatNP-TEFb complex is refined at 2.1 A resolutionto an R of 21.9% and Rfree of 26.4%, and the structure of the TatNP-TEFbNATP is refined at 3.0 A resolution to an R of 21.8% and Rfree of28.6%. The Cdk9 subunit is phosphorylated at Thr 186 and is in aconformation characteristic for active cyclin-dependent kinases18.

The overall structure of the TatNP-TEFbNATP complex depicted inFig. 1 shows that Tat acquires an extended conformation mainlythrough interactions with cyclin T1 (see Supplementary Fig. 1 forthe definition of secondary structure elements). As expected fromearlier biochemical studies19, Tat forms a Zn-mediated bridge withCys 261 of cyclin T1. Tat binding to P-TEFb buries 3,499 A2 of surfacearea, of which 88% is on cyclin T1 and 12% is on Cdk9. Clearly, Tathas evolved to bind tightly to P-TEFb as 37% of its folded portion(amino acids 1–49) surface is complementary to the kinase. Theamount of surface buried between P-TEFb and Tat is double theaverage value for stable protein–protein interactions20. The CDK–cyclin interaction surface is smaller in P-TEFb than in other suchcomplexes21. However, Tat augments it by inserting in a groove at theheterodimer interface and leading to a more stable and more activeP-TEFb complex17.

Tat is encoded by two exons. Amino acids 1–72 encoded by thefirst exon are highly conserved and comprise five regions (Fig. 2a)that are sufficient for transactivation of the viral LTR promoter. Thefirst three regions constitute a minimal activation domain capable ofbinding to cyclin T1. Indeed, the residues 1–49 are ordered in thecrystal structure of TatNP-TEFb, whereas the density for the rest ofTat (residues 50–86) is not defined.

1Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-7696, USA. 2Biochemistry Department, University ofIowa, Iowa City, Iowa 52242, USA.

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The acidic/proline-rich region is in an extended loop conforma-tion and folded as a random coil with two type II b turns and one typeII9 b turn (Fig. 2b, c). The mutations of the acidic or proline residuesas well as deletion mutants within this region impair the activity ofTat22. A cysteine-rich region and core regions form a more compactstructure with a random coil, two helices, a tail, and two bound Znions (Fig. 2b, c). The short 310-helix is located between the Zn ions(Zn1 and Zn2) and interacts with both zincs. The a-helix interactsonly with Zn1 and extends towards the carboxy terminus of theactivation domain. Zn1 is coordinated by Cys 22, His 33, Cys 34and Cys 37 of Tat (Fig. 2d, e), and Zn2, located 10.7 A away, is coor-dinated by Cys 25, Cys 27 and Cys 30 of Tat and Cys 261 of cyclin T1(Fig. 2f, g). The observed coordination of Zn ions explains earliermutational studies pointing to the importance of His 33 and allcysteines, except Cys 31, in the Zn-dependent function of Tat23,24.The Cys 31 is replaced by a serine in several Tat variants and thisreplacement does not diminish the protein function25. The zinc fingers(ZnFs) in Tat should be considered unique because protein foldsearches did not uncover similar ZnF motifs in any other protein.

In addition to the main-chain hydrogen bonds involved in secondarystructure elements, only six intramolecular hydrogen bonds were foundin Tat (Supplementary Fig. 2). Three of these hydrogen bonds werebetween the side chain of Lys 41 and main-chain oxygens of Thr 23,Cys 25 and Cys 30. The latter intramolecular interactions seem to becrucial for the structural integrity of the cysteine-rich/core modulebecause mutation of Lys 41 impairs the activity of Tat25,26.

Recently the crystal structure of the fusion of equine cyclin T1 andequine infectious anaemia virus (EIAV) Tat in complex with EIAV

TAR RNA was reported27. The basic RNA-recognition motif of EIAVTat adopts a helical structure whose flanking regions interact with anequine cyclin T1 and both proteins coordinate the stem-loop struc-ture of EIAV TAR27. However, the key residues in EIAV TAT andequine cyclin T1 involved in ternary complex formation with TARRNA, are different in HIV-1 Tat and human cyclin T1 (refs 27–29).The TAR RNAs from HIV-1 and EIAV have different structures aswell27,30. Moreover, the photocross-linking and protein footprintingstudies31 show that HIV-1 TAR RNA loop interacts with cyclin T1residues 252–260, which is far from the EIAV TAR RNA docking site.These differences make it difficult to model HIV-1 TAR binding tothe TatNP-TEFb complex on the basis of the structure of the EIAVcomplex27. However, it is appropriate to apply the structure of HIV-1TatNP-TEFb to model the portion of the activation domain for theEIAV Tat sharing high sequence identity with HIV-1 Tat in ZnF andcore regions (Supplementary Fig. 3a). The proposed model of EIAVTat ZnF and core regions in complex with cyclin T1 is shown inSupplementary Fig. 3b and displays an uninterrupted connectionwith the basic region. The high sequence conservation of the centralZnF-core motif in Tat proteins, compared with poorly conservedflanking regions, indicates that the binding of ZnF-core motif to acyclin T1 surface is the most conserved structural feature sharedamong Tat transcriptional regulators.

TatNP-TEFb interactions

Interactions between Tat and P-TEFb can be divided into four areas(Fig. 3). The first area involves a U-shaped, acidic/proline-rich regionof Tat and a wide depression between the two cyclin repeats of cyclinT1. The second area involves a b-turn in the acidic/proline-rich regionof Tat and the T-loop of Cdk9. In the third area, the cysteine-richregion and a core region of Tat interact with a depression betweenthe cyclin repeats of cyclin T1 and a point close to the helix H59. Thefourth area involves the second ZnF of Tat and the Cys 261 of cyclin T1.

Tat possesses an extended hydrophobic patch comprised of theamino-terminal methionine and amino acid residues from a cysteine-rich region and the core region that are packed against the hydrophobic

T-loop

Cdk9 Cyclin T1

Tat

C

N

ATP

aNC

N

b

Figure 1 | Overall structure of the TatNP-TEFbNATP. a, b, Ribbon representa-tion (a) and surface representation (b) of the TatNP-TEFbNATP structure. Cdk9is light orange, cyclin T1 is pale green and Tat is magenta. The side chains of theCdk9-interacting residues of Tat, Cys 261 of cyclin T1 and ATP analogue aredrawn as sticks, and the zinc and magnesium atoms are drawn as cyan and lightblue spheres, respectively. The dashed lines represent the missing link betweenLys 88 and Gly 97 of Cdk9, and between Leu 252 and Cys 261 of cyclin T1.

Exon 2Glutamine-richCoreCysteine-rich/ZnFAcidic/proline-rich Basic1 86–101

Activation domain RNA-binding domainnuclear localization signal

90°

90°

H33

C34

C22

C37

Zn1C30

C27C25

C261CycT1

Zn2

K29

a

b

c

d e f g

7257484020

Figure 2 | Structure of P-TEFb-bound Tat. a, Diagram of the regions andfunctional domains of HIV-1 Tat proteins. b, c, Stick (b) andcartoon(c) representations of the Tat structure in two orientations. The regionsof Tat are coloured as in a. A close-up view of Zn1 and Zn2 coordination and aportion of 2Fo2Fc Fourier maps (mesh) contoured at 1s are shown (d–g).

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surface of cyclin T1 (Supplementary Fig. 4a). Moreover, several inter-molecular van der Waals contacts can be seen at discrete locations(Supplementary Fig. 4b, c).

Tat forms numerous direct and water-mediated hydrogen bondswith both Cdk9 and cyclin T1 (Supplementary Table 1 and Sup-plementary Figs 5 and 6). The fewer Tat-stabilizing intramolecularhydrogen bonds compared with intermolecular TatNP-TEFb hydro-gen bonds indicate that overall the conformation of Tat is deter-mined predominantly by its interactions with P-TEFb.

Conformational changes of Tat and P-TEFb

Several solution structures of Tat variants have been reported32. In theabsence of interacting partners, Tat does not have prominent secondarystructure elements, indicating the flexible nature of free Tat proteins32.However, upon interaction with P-TEFb and two Zn ions, the activa-tion domain of Tat acquires a well ordered structure (Fig. 1).

To elucidate Tat-induced conformational changes in P-TEFb, wecompared the structure of the TatNP-TEFbNATP complex with thestructure of P-TEFbNATP (PDB ID 3blq) solved previously21.Although there are differences in P-TEFb subunit constructs andcrystal packing, four notable changes were observed. First, Tat bind-ing resulted in the unfolding and disordering of the cyclin T1 a-helixHC (residues 253–256) and a 4.6 A displacement of the C-terminalportion of the preceding a-helix H59 (Fig. 4a). These changes exposea buried surface in cyclin T1 that is used for the binding of the ZnFand core region sequences of Tat. Second, when the cyclin T1 sub-units of P-TEFbNATP and TatNP-TEFbNATP were aligned, a signifi-cant, 8.5u rotation was detected for the Cdk9 subunit (Fig. 4b). Third,

the shift of phospho-Thr 186 by 2.6 A resulted in the formation oftwo hydrogen bonds with Arg 65 and one additional hydrogen bondwith Arg 148 (Fig. 4c). Fourth, the concerted allosteric changes resultin the switch of the conformations in the b1b2-loop and b3aC-loopthat are in close proximity to the ATP binding site (Fig. 4d). Tounderstand the possible sequence of events leading to the latter con-formational changes upon Tat binding, we superimposed the Cdk9subunit of TatNP-TEFbNATP with the Cdk9 subunit of P-TEFbNATP.Figure 4d demonstrates a significant shift of the first cyclin repeat ofcyclin T1 towards Cdk9 in the structure of TatNP-TEFbNATP relativeto structure of P-TEFbNATP. This shift reaches up to 4.8 A and leadsto steric hindrance between the a-helix H5 of cyclin T1 and the b3aC-loop of Cdk9, in particular between Val 134 and Ser 138 of cyclin T1and Gly 58 and Met 52 of Cdk9, respectively (Fig. 4e). To release thissteric hindrance, the Gly 58 of Cdk9 is shifted 2.4 A away fromVal 134 of cyclin T1 and the part of the Cdk9 b3aC-loop comprisedof residues 51–55 changed their conformation. As result of this con-formational change the Leu 51 clashes with Phe 30, pushing it awayand forcing the residues 25–32 of b1b2-loop to acquire a conforma-tion that is different than that in P-TEFbNATP (Fig. 4e). The Tat-induced conformational changes in Cdk9 modify the substrate-bindingsurface of P-TEFbNATP. This provides a structure-based explanationfor the earlier findings showing that Tat-bound P-TEFb phosphory-lates not only the Ser 2 but also the Ser 5 in the heptad repeats of theRNA polymerase II large subunit C-terminal domain17.

Structural constraints for Tat mutations

HIV-1 genes are prone to rapid mutation leading to high geneticvariability. HIV-1 has been classified into three genetic groups: major(M), outlier (O) and new (N) that are non-M and non-O33. MostHIV-1 infections are caused by group M viruses. The group M isdivided into nine subtypes: A–D, F–H, J and K33. Genetic variationwithin a subtype is 15–20%, and is 25–35% between subtypes33. In

II

I

III

IV

Cdk9

Cyclin T1

b

a

Figure 3 | The Tat-interacting areas of P-TEFb. a, b, Mapping of the Tat-interacting residues of P-TEFb (a) to the surface of P-TEFb and (b) to theribbon diagram of P-TEFb. The subunits and interacting areas are colouredaccording to definitions shown in a.

R65

R172R148

Phospho-T186

H5′HC

8.5°

β1β2-loop

H5

S138

V134

F30

L51

M52

G58

β1β2-loop

β3αC-loop

e

a

c

b

d

Figure 4 | Comparison of the crystal structures of P-TEFbNATP and TatNP-TEFbNATP. a, Shift of H59 and disordering of HC in cyclin T1 of TatNP-TEFbNATP (orange). b, Superimposition of cyclin T1 showing rotation ofCdk9. P-TEFbNATP: Cdk9 (green), cyclin T1 (blue); TatNP-TEFbNATP: Cdk9(salmon), cyclin T1 (cyan). c, Interactions of phospho-Thr 186 in panelb. P-TEFbNATP (green); TatNP-TEFbNATP (coloured by atom type).d, Superimposition of Cdk9 showing the shift of cyclin T1. e, Concertedmovement of the cyclin T1 H5 and the b3aC-loop of Cdk9 causing theconformational switch of the Cdk9 b1b2-loop upon Tat binding to P-TEFb.The residues causing sterical hindrance are drawn as sticks. Colours in d ande are as in b.

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line with this observation, Tat tolerates up to 40% sequence variationwithout loss of transcriptional activity32. Our structure providesinsight into how mutations in Tat are tolerated. To facilitate thedescription of sequence variations, we produced sequence logos34

of the Tat activation domain using the alignment of 1,118 Tatsequences35 (Fig. 5a). The alignment indicates that half of the residueshave a higher degree of conservation, with 14 invariant residues and10 residues with randomly occurring polymorphs (Fig. 5b). Theapparent reasons for the conservation are the following: involvementin Zn coordination, interaction with the T-loop of Cdk9, stabiliza-tion of Tat structure by intramolecular hydrogen bonds, interactionwith a hydrophobic surface of cyclin T1, formation of hydrogenbonds with cyclin T1, and providing rigidity to the loop connectingthe acidic/proline-rich region and a cysteine-rich region. There is noalternative for glycine at position 15, because any mutation wouldcreate a backlash with Asn 53 side chain of cyclin T1 and disrupt itshydrogen bond with Gln 17 of Tat (Supplementary Fig. 7). The ana-lysis of TatNP-TEFb structure alone does not provide an immediateexplanation for the functional importance of Lys 28. However, itexplains an earlier finding indicating that acetylated Lys 28 interactswith Asn 257 of cyclin T1 and facilitates the formation of TatNTARNP-TEFb complex36. Indeed, Lys 28 is located at the surface of the secondZnF (Fig. 2f); in close proximity to the missing cyclin T1 residues(252-260) linking Cys 261 to the cyclin domain and harbouringAsn 257. Acetylated Lys 28 could reach the Asn 257 side chain andform an intermolecular hydrogen bond.

Residues with a high mutation rate are divided into two groups:those with predominantly functionally equivalent substitutions(Fig. 5c) and those with a variety of substitutions (Fig. 5d). All theseresidues, except Gln 35 and Ile 39, are fully or partially exposed. Noneof the observed mutations would disrupt interactions of Tat withP-TEFb. Gln 35 penetrates deeply into a depression on cyclin T1and forms a hydrogen bond with Asn180 (Supplementary Fig. 7a).All Gln 35 mutations (Fig. 5a), except tyrosine, could be accommo-dated with minor adjustments in the positions of surrounding sidechains. However, modelling of tyrosine shows that it may shift theN-terminal methionine without disrupting the TatNP-TEFb (Sup-plementary Fig. 7a). Another frequently mutated residue buriedat the TatNP-TEFb interface is Ile 39 (Fig. 5a). The Ile 39 side chainfaces a groove that is occupied by a water molecule (SupplementaryFig. 7c), and hence all Ile 39 mutations could be easily accommodatedwithout disrupting the TatNP-TEFb.

Implications for P-TEFb activation

The TatNP-TEFb structure provides insight into how Tat functions inrecruiting P-TEFb to the HIV LTR. The first step of this processinvolves a Tat-induced release of P-TEFb from the 7SK snRNP-containing complex13,37. Most of P-TEFb within the cell is held in aninactive form by HEXIM1, which is bound to 7SK snRNA along withother proteins38,39. Several experiments show that inhibition ofP-TEFb by HEXIM1 resembles the inhibition of Cdk2NcyclinA byp27kip1 (ref. 40). In the Cdk2NcyclinANp27kip1 crystal structure the sidechains of Phe 87 and Tyr 88 of p27kip1 enter the active site cleft andprevent the binding of ATP40. Mutational studies of invariant tyrosineand phenylalanine residues of HEXIM1 showed that Tyr 271 andPhe 208 mutations relieved the inhibitory activity of P-TEFb withoutdissociation of HEXIM1 (ref. 41). These results indicate that HEXIM1blocks the binding of ATP to Cdk9 in a manner similar to p27kip1. Insuch a case the flipping of the b1b2-loop conformation would lead todissociation from P-TEFb (Supplementary Fig. 8). In support of thisidea, P-TEFb with a shorter cyclin T1 (1–254) and a missing a-helixHC was not capable of binding 7SK snRNA42, indicating the import-ant contribution of this helix to P-TEFbNHEXIM1N7SK snRNA com-plex formation. Because Tat unfolds the HC of cyclin T1 this also mayfacilitate the release of HEXIM1 (Supplementary Fig. 8).

Conclusions

The emergence of multidrug resistant HIV-1 strains requires thedevelopment of new drugs capable of stopping viral replication43.Tat has a major role in the viral replication and remains a viabletarget for crystallographic studies and structure-based drug design.The structure of TatNP-TEFb described here revealed a large inter-action interface buried between these two factors and showed howlocal conformational changes caused by Tat mutations could be tol-erated without disruption of the complex. The extended and flexibleinteraction-dependent conformation of Tat suggests that designingcompounds that would block the interaction of Tat with P-TEFbwould be a challenging task. The disadvantage of such inhibitorsmight be also the interference with the function of cellular factors(for example HEXIM1, BRD4, etc.) that bind to and are essential forthe regulation of P-TEFb. On the other hand, the Tat binding changesthe overall shape and surface of P-TEFb. This provides an opportun-ity for the design of inhibitors that would not bind to or inhibit thenormal cellular function of P-TEFb, but would be specific only to theform of P-TEFb used by the virus.

G48

M1

D5

P6

P10

W11 P14

G15S16

P18

T20

C22

C25C27

K28

H33C34

F38C30

C37

K41L43

I45G44

A42

E2

V4

L8

H13

Y26

C31

F32

P3

R7E9K12

Q17

K19

A21

T23

N24

K29

Q35V36

Y47 T40

S46

I39

a

b c d

Bits

Figure 5 | A conservation-dependent location ofTat’s amino acid residues. a, Sequence logos ofthe Tat activation domain. b, Location of Tatinvariant residues (yellow) and residues withrandomly occurring polymorphs (deep salmon);c, residues with predominantly functionallyequivalent substitutions (green) and d, residueswith a variety of substitutions (magenta). Inpanels b–d Tat is drawn as grey ribbon, and cyclinT1 (pale cyan) and Cdk9 (pale green) are shownin surface representation. The highlighted Tatresidues and Cys 261 of cyclin T1 are representedas ball-and-stick diagrams. The zinc atoms areshown as large grey spheres.

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METHODS SUMMARYC-terminally His-tagged human Cdk9 (1–345), human cyclin T1 (1–266) and

HIV-1 Tat (1–86) were co-expressed in insect cells and purified by Ni-NTA and

Mono S columns. The purified TatNP-TEFb complex was crystallized using the

sitting-drop vapour-diffusion method. The TatNP-TEFbNATP crystals were

obtained by soaking AMP-PNP and magnesium chloride into the TatNP-TEFb

crystals. Transformation of crystals to a low-humidity form markedly improved

the diffraction enabling the complete data collection at high resolution using

synchrotron radiation. The TatNP-TEFb structure was solved using molecular

replacement method and refined at 2.1 A resolution. The positions of AMP-PNP

and magnesium in TatNP-TEFbNATP structure were determined by the differ-

ence Fourier map and the structure was refined at 3 A resolution. Both structures

exhibited electron density maps of high quality and provided the models with

excellent geometry.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 21 January; accepted 27 April 2010.

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32. Campbell,G.R.&Loret,E.P.Whatdoesthestructure-functionrelationshipoftheHIV-1Tat protein teach us about developing an AIDS vaccine? Retrovirology 6, 50 (2009).

33. Hemelaar, J., Gouws, E., Ghys, P. D. & Osmanov, S. Global and regionaldistribution of HIV-1 genetic subtypes and recombinants in 2004. AIDS 20,W13–W23 (2006).

34. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logogenerator. Genome Res. 14, 1188–1190 (2004).

35. Kuiken, C. et al. HIV Sequence Compendium 2009, LA-UR 09–03280 (TheoreticalBiology and Biophysics Group, Los Alamos National Laboratory, USA) (2009).

36. D’Orso, I. & Frankel, A. D. Tat acetylation modulates assembly of a viral-host RNA-protein transcription complex. Proc. Natl Acad. Sci. USA 106, 3101–3106 (2009).

37. Barboric, M. et al. Tat competes with HEXIM1 to increase the active pool ofP-TEFb for HIV-1 transcription. Nucleic Acids Res. 35, 2003–2012 (2007).

38. Michels, A. A. et al. MAQ1 and 7SK RNA interact with CDK9/cyclin T complexesin a transcription-dependent manner. Mol. Cell. Biol. 23, 4859–4869 (2003).

39. Yik, J. H. et al. Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymeraseII transcription by the coordinated actions of HEXIM1 and 7SK snRNA. Mol. Cell 12,971–982 (2003).

40. Russo, A. A., Jeffrey, P. D., Patten, A. K., Massague, J. & Pavletich, N. P. Crystalstructure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclinA-Cdk2 complex. Nature 382, 325–331 (1996).

41. Li, Q. et al. Analysis of the large inactive P-TEFb complex indicates that it contains one7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containingCdk9 phosphorylated at threonine 186. J. Biol. Chem. 280, 28819–28826 (2005).

42. Chen, R., Yang, Z. & Zhou, Q. Phosphorylated positive transcription elongationfactor b (P-TEFb) is tagged for inhibition through association with 7SK snRNA.J. Biol. Chem. 279, 4153–4160 (2004).

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank J. Lovelace and G. E. Borgstahl for maintenance andmanagement of the Eppley Institute’s X-ray Crystallography facility;D. G. Vassylyev for the zonal scaling instruction files. This work is supported by theNIH grants GM35500 and AI074392 to D.H.P., by Nebraska Department of Healthand Human Services grant LB506 and in part by NIH grant GM082923 to T.H.T.This work is also based on research conducted at the Advanced Photon Source onthe Northeastern Collaborative Access Team beamlines, which are supported byaward RR-15301 from the National Center for Research Resources at the NationalInstitutes of Health. Use of the Advanced Photon Source is supported by the USDepartment of Energy, Office of Basic Energy Sciences, under Contract No.DE-AC02-06CH11357. The Eppley Institute’s X-ray Crystallography facility issupported by Cancer Center Support Grant P30CA036727.

Author Contributions T.H.T. managed the crystallization and structuredetermination part of the project, solved the crystal structures and prepared themanuscript. N.D.B. obtained the crystals. Diffraction data collection wasperformed by N.D.B. and T.H.T. Protein cloning, expression, purification and writingthe corresponding methods sections were performed by S.C.S., K.V. and J.J.C.,respectively. D.H.P. managed the protein production part of the project, and helpedgenerate and edit the manuscript.

Author Information Atomic coordinates and structure factors for TatNP-TEFb andTatNP-TEFbNATP structures have been deposited in the Protein Data Bank withaccession numbers 3mi9 and 3mia, respectively. Reprints and permissionsinformation is available at www.nature.com/reprints. The authors declare nocompeting financial interests. Readers are welcome to comment on the onlineversion of this article at www.nature.com/nature. Correspondence and requestsfor materials should be addressed to T.H.T. (ttahirov@unmc.edu).

NATURE | Vol 465 | 10 June 2010 ARTICLES

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METHODSExpression of TatNP-TEFb complex. Sf9 insect cells were co-infected with

three baculoviruses that individually directed the expression of C-terminally

His-tagged human Cdk9 (1–345), human cyclin T1 (1–266) and HIV-1 Tat

(1–86). The three coding sequences were individually cloned into the pENTR/

SD/D-TOPO vector (Invitrogen) after PCR amplification.

The baculoviruses were generated using BaculoDirect C-Term Expression kit

(Invitrogen), and these viruses were transfected into Sf9 cells following the

manufacturer’s instructions. The viruses were amplified several times to obtain

viral stocks with 108 plaque-forming units per millilitre. Stocks were titred by

infecting individual wells of a six-well plate containing 1 3 106 Sf9 cells with

0.001, 0.01, 0.1, 1, 10 or 100ml of the stocks. After 48 h the cells were fixed with

3.7% formaldehyde for 15 min and stained using first a gp64 antibody

(Novagen), then by an alkaline phosphatase-coupled goat anti-mouse IgG

(Sigma-Aldrich); colour development with 5-bromo-4-chloro-39-indolyphosphate

p-toluidine Salt (BCIP) and Nitro-Blue tetrazolium chloride (NBT) (Sigma-

Aldrich) followed. The presence of the gene of interest was determined by conduct-

ing PCR on DNA isolated by phenol extraction from the stocks.

Large scale production of the TatNP-TEFb complex was carried out using

baffled 2 l glass Erlenmeyer flasks containing 1 l Sf-900 serum-free medium

(GIBCO) with 1.5 3 106 Sf9 cells/ml. The flasks were incubated at 70 r.p.m. in

a rotary shaker at 27uC. Typically, 6 l of cells were infected at once by adding

20 ml of each of the three viral stocks to each litre of culture. The flasks were then

incubated for an additional 72 h.

Purification of the TatNP-TEFb complex. To avoid protein degradation, all

solutions and equipment were kept cold and the purification was performed

swiftly. Cells were collected from six 1 l cultures by spinning for 10 min at

4,200 r.p.m. in a Beckman JS-4.2 rotor (3,600g). Each of the six pellets was

resuspended in 20 ml of PBS supplemented with 1 mg ml21 E-64 protease inhi-

bitor (Calbiochem), 13 Roche Complete Protease Inhibitor (Roche), and 0.1%

of a saturated phenylmethylsulphonyl fluoride (PMSF) solution in isopropanol

then transferred to 40 ml tubes. After centrifuging at 3,000 r.p.m. for 5 min in a

Beckman JS 13.1 rotor (1,000g), the supernatants were decanted and pellets were

resuspended in 12 ml of lysis buffer (5 mM imidazole, 150 mM NaCl, 10 mM Tris

pH 7.8, 1% Triton, 2 mM MgCl2, 1mg ml21 E-64, 13 Roche Complete Protease

Inhibitor, and 0.1% PMSF solution). Lysates were combined into three 50 ml

plastic beakers on ice, and each was sonicated five times for 15 s with a 5-min

pause between bursts. Cell lysates were combined and brought to 500 mM NaCl

by adding 5 M NaCl and mixing vigorously then centrifuged at 45,000 r.p.m. for

45 min in a Beckman 55.2Ti rotor (167,000g) in Oak Ridge tubes. The high-speed

supernatant was incubated with 4.5 ml of Qiagen Ni-NTA agarose that had been

equilibrated in lysis buffer containing 500 mM NaCl. After 60 min the resin was

pelleted at 1,200 r.p.m. for 3 min in a Beckman JS-13.1 rotor (146g). The resin

was resuspended in wash buffer (5 mM imidazole, 500 mM NaCl, 10 mM Tris

pH 7.8, 1% Triton, and 0.1% PMSF solution) and transferred equally into three

20 ml Bio-Rad disposable columns. Each column was washed successively with

10 ml of wash buffer, 15 ml of HSW (25 mM imidazole, 1 M NaCl, 10 mM Tris

pH 7.8, 1% Triton, and 0.1% PMSF solution), and 15 ml of 70 mM HGKE wash

(25 mM imidazole, 70 mM KCl, 25 mM HEPES pH 7.6, 15% glycerol, 0.1 mM

EDTA, 1% Triton, and 0.1% PMSF solution). Each column was then eluted with

6 ml of HGKE wash supplemented with 300 mM imidazole. The eluted material

was pooled and centrifuged at 12,500 r.p.m. for 15 min in a Beckman JS-13.1

rotor (16,000g) and then loaded onto a 1 ml Mono S column that had been

equilibrated in 70 mM HK buffer (70 mM KCl, 10 mM HEPES pH 7.6, 1 mM

dithiothreitol (DTT), and 0.1% PMSF solution). After a 5 ml wash the column

was eluted with a 17.5 ml gradient from 70 to 620 mM KCl in HK buffer

(Supplementary Fig. 9a). Free P-TEFb eluted at 220 mM KCl and the TatNP-

TEFb complex at 420 mM KCl. The peak fractions were relatively free of con-

taminating proteins (Supplementary Fig. 9b). The purification protocol was

performed more than 20 times with minor modifications and usually resulted

in 5–10 mg of the TatNP-TEFb complex per 6 l of cells. The samples obtained

were kept in small aliquots at 280 uC.

Crystallization. The frozen TatNP-TEFb samples were thawed on ice, dialysed

against 400 mM KCl, 10 mM HEPES buffer (pH 7.5), and 2 mM tris(2-

carboxyethyl)phosphine hydrochloride (TCEP), then concentrated to

5–6 mg ml21 using Millipore Ultrafree centrifugal devices. Only few fractions

reached these concentrations because sudden precipitation occurred on most

fractions even at lower concentrations. The state of sample aggregation was

monitored by dynamic light scattering. Application of the solubility screening

protocol described previously44 did not improve the sample solubility and

aggregation. Initial crystallization screening was performed at a temperature

of 295 K in 96-well plates using 50% diluted Crystal Screen and Crystal Screen

2 solutions (Hampton Research) by the sitting-drop vapour-diffusion method

by mixing 1 ml of complex solution with 1 ml of reservoir solution. TCEP (1 mM)

was added to each condition and finally 400 mM KCl was added to each

reservoir. Crystals appeared in 37th condition of the Crystal Screen 2 containing

50 mM HEPES buffer (pH 7.5), 5% PEG 8,000, 2 mM TCEP, and 2% ethylene

glycol in the crystallization drop plus 400 mM KCl in the reservoir only.

Variations in concentration of the components, molecular mass of PEGs, buffers

and pH, and inclusion of additives from Hampton Research Additive Screen

revealed the following conditions producing crystals of better shape: 50 mM

HEPES buffer (pH 7.5), 4.25–5% PEG 20,000, 1 mM TCEP, and 20 mM glycyl-

glycyl-glycine mixed with protein solution. The reservoir solution contained

also 400 mM KCl. Most drops produced small needle-like crystals in 1–2 days;

however, in some of remaining clear drops larger crystals of hexagonal prism

shape (Supplementary Fig. 10a) appeared in 1–3 weeks and sometimes after

1–3 months. The growth of crystals normally continued for 2–4 weeks, reaching

the maximum dimensions of 0.25 3 0.25 3 1.5 mm3. The polarization of light by

these crystals was barely detectable. The SDS–PAGE of dissolved crystals showed

all three subunits of TatNP-TEFb in stoichiometric (1:1:1) proportions

(Supplementary Fig. 10b). The TatNP-TEFbNATP crystals were obtained by soak-

ing the TatNP-TEFb crystals in crystallization solution with 1 mM ATP analogue

AMP-PNP and 5 mM magnesium chloride for 40 min.

X-ray diffraction data collection and processing. For the preliminary studies

the crystals were soaked in cryoprotectant for a few seconds, scooped with a

nylon-fibre loop and flash-cooled in a dry nitrogen stream at 100 K. The best

cryoprotectant was obtained by adding 25% (v/v) glycerol or ethylene glycol to

the reservoir solution. Preliminary diffraction data were collected at cryogenic

temperature on a Rigaku R-AXIS IV imaging plate using Osmic VariMax HR

mirror-focused CuKa radiation from a Rigaku FR-E rotating anode operated at

45 kV and 45 mA. Despite a large volume of crystals, the diffraction was poor for

most crystals, reaching a resolution of only 4.2–4.5 A. Occasionally some crystals

diffracted better, up to a resolution of 3.5–3.8 A. Inspection of unit cell para-

meters showed that, in general, better-diffracting crystals had slightly shorter

(1–1.5 A) parameters a and b. Because the crystals with such properties are often

capable of transformation to a low-humidity form, we started such experiments

with TatNP-TEFb crystals with modification of earlier described experimental

setup45. Instead of mounting the crystal directly in capillary, we mounted the

cryoprotectant-soaked crystal (with the content of glycerol or ethylene glycol

reduced to 12–16% v/v) in a MiTeGen MicroMeshes, wiped the excess but not all

solution around the crystal with the fine-cut filter paper over the top of the well

with cryoprotectant solution, immediately placed it in a capillary having a

cryoprotectant solution on the wider end and sealed its bottom touching the

goniometer base (without using the hot wax). Cryoprotectant in capillary was

replaced with solutions each having the PEG 20,000 concentration increased by

3% every 5–10 min until initial signs of transformation such as the reduction of

the volume of the remaining solution around the crystal. Then the capillary was

removed and the crystal was flash-cooled under the stream of nitrogen gas. It was

important not to reach the point of transformation when an apparent ‘sweating’

of crystal occurs because further reduction of the relative humidity results in

marked shrinkage of crystals, especially along the axis c, and complete loss of

diffraction. This method of transformation to a low-humidity form was effective

only for crystals having cross section of over 0.15 mm, probably due to fast

dehydration of smaller crystals. The most successful transformation of TatNP-

TEFb crystals was accompanied with the reduction of unit cell parameters a and c

by 4.5 A and 1.5 A, respectively, and extension of the X-ray diffraction resolution

limits from 4.5–3.5 A to 2.1 A. The high-resolution diffraction data were col-

lected using synchrotron radiation at the Advanced Photon Source on the

Northeastern Collaborative Access Team beamlines BL24ID-C and BL24ID-E.

To minimize the radiation damage, complete data sets were obtained from one

crystal of TatNP-TEFb and one crystal of TatNP-TEFbNATP, each exposed at six

positions. The data set for the calculation of the Zn anomalous diffraction signal

was collected by exposing only one position of the crystal with a beam intensity

reduced to 20%. The intensity data were indexed, integrated, and scaled using the

HKL2000 program package46 (Supplementary Table 2).

Structure determination and refinement. The structure was determined by the

molecular replacement method starting with the coordinates of P-TEFb (PDB

entry 3blh)21. All amino acid residues with temperature factors above 80 A2 were

excluded from the positioned model before the calculation of phases. Density

modification with CNS software47 reduced the model bias and improved the

electron density maps. The building of the Tat structure and missing parts of the

Cdk9 and cyclin T1 subunits, as well as adjustment of the initial model, was

performed manually in five rounds with TURBO-FRODO software. The posi-

tions of Zn atoms were confirmed using anomalous difference Fourier map. The

refinement of the completed TatNP-TEFb structure was performed using con-

jugate gradient minimization, simulated annealing, and temperature factor

refinement CNS protocols47. Finally, 341 water molecules were added with the

doi:10.1038/nature09131

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water-pick protocol of CNS. Application of zonal scaling48 and bulk solventcorrection improved the geometry of the model and the quality of the maps

(Supplementary Fig. 11). Density was not observed for residues 1–7, 89–96, 345

and the C-terminal His-tag of Cdk9, for residues 1–6, 253–260 and 262–266 of

cyclin T1, and for residues 50–86 of Tat. The broadened density and high tem-

perature factor for Cys 261 illustrate its high mobility or disorder. The partial

occupancy of the Cys 261 position by a histidine from a disordered portion of Tat

or from a disordered His-tag of neighbouring molecule cannot be excluded. The

refined structure of TatNP-TEFb was used to determine the structure of TatNP-

TEFbNATP. The difference Fourier map revealed the site of AMP-PNP and

magnesium binding (Supplementary Fig. 12). The refinement statistics for both

structures are provided in Supplementary Table 2. The electron density for the

Val 190 and Arg 284 of Cdk9, only residues located in the disallowed region of

Ramachandran plot, is well defined. The unfavourable main-chain conforma-

tions of Val 190 and Arg 284 are stabilized by intramolecular hydrogen bonds. All

figures displaying the protein structures were prepared with PyMol software

from Delano Scientific.

44. Jancarik, J., Pufan, R., Hong, C., Kim, S. H. & Kim, R. Optimum solubility (OS)screening: an efficient method to optimize buffer conditions for homogeneity andcrystallization of proteins. Acta Crystallogr. D 60, 1670–1673 (2004).

45. Tahirov, T. H. et al. High-resolution crystals of methionine aminopeptidase fromPyrococcus furiosus obtained by water-mediated transformation. J. Struct. Biol.121, 68–72 (1998).

46. Otwinowski, Z. & Minor, W. Processing of X-ray Diffraction Data Collected inOscillation Mode (eds. Carter, J. C. W. & Sweet, R. M.) (1997).

47. Brunger, A. T. et al. Crystallography & NMR system: A new software suite formacromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

48. Vassylyev, D. G., Vassylyeva, M. N., Perederina, A., Tahirov, T. H. & Artsimovitch,I. Structural basis for transcription elongation by bacterial RNA polymerase.Nature 448, 157–162 (2007).

doi:10.1038/nature09131

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