The Crystal Structure of Human CDK7 and Its Protein Recognition Properties

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Graziano Lolli, Edward D. Lowe, Nick R. Brown, ylation has not been identified, but in vitro active phos- and Louise N. Johnson* pho-CDK2/cyclin A will promote phosphorylation and Laboratory of Molecular Biophysics CDK7/cyclin H assembly (Fisher et al., 1995). Department of Biochemistry CDK7 also plays a central role in the regulation of the University of Oxford initiation phase of messenger RNA synthesis by RNA Rex Richards Building pol II. CDK7 is part of the general transcription factor Oxford, OX1 3QU TFIIH (Feaver et al., 1994; Roy et al., 1994; Schiekhattar United Kingdom et al., 1995; Serizawa et al., 1995) and in this complex phosphorylates RNA polymerase II (RNA pol II) large subunit C-terminal domain (CTD) (Goodrich and Tjian, Summary 1994; Lu et al., 1992; Oelgeschlager, 2002; Palancade and Bensaude, 2003; Shilatifard et al., 2003). The CTD CDK7, a member of the cyclin-dependent protein ki- contains a tandem array of 52 repeats (in humans) with nase family, regulates the activities of other CDKs the consensus sequence YSPTSPS (using the single through phosphorylation on their activation segment letter code). CDK7 preferentially phosphorylates Ser5 in and hence contributes to control of the eukaryotic cell this sequence. Phosphorylation of the CTD facilitates cycle. CDK7 also assists in the regulation of transcrip- promoter clearance, initiation of transcription (Dahmus, tion as part of the transcription factor TFIIH complex. 1996), and recognition by RNA processing enzymes. For maximum activity and stability, CDK7 requires In order to form a stable dimeric active complex with phosphorylation, association with cyclin H, and asso- cyclin H, CDK7 must be phosphorylated on a conserved ciation with a third protein, MAT1. We have determined threonine (T170 in the human protein) in the activation the crystal structure of human CDK7 in complex with loop (Fisher et al., 1995; Martinez et al., 1997). Unlike ATP at 3 A˚ resolution. The kinase is in the inactive other CDKs, CDK7 has an additional phosphorylation conformation, similar to that observed for inactive site in the activation segment (Ser164 in the human pro- CDK2. The activation segment is phosphorylated at tein). Phosphorylation at this site enhances activity and Thr170 and is in a defined conformation that differs cyclin binding (Martinez et al., 1997). The trimeric CDK7/ from that in phospho-CDK2 and phospho-CDK2/cyclin cyclin H/MAT1 complex is active in the absence of phos- A. The functional properties of the enzyme against phorylation but phosphorylation stabilizes the complex CDK2 and CTD as substrates are characterized through and stimulates activity toward the CTD of RNA Pol II kinase assays. Experiments confirm that CDK7 is not without affecting activity on CDK2 (Larochelle et al., a substrate for kinase-associated phosphatase. 2001). Additional factors also regulate CDK7 substrate selectivity in TFIIH (Akoulitchev et al., 2000; Akoulitchev and Reinberg, 1998; Chen et al., 2003; Nishiwaki et al., Introduction 2000; Serizawa, 1998). Unlike other cell cycle CDKs, cyclin H levels and CDK7 The cyclin-dependent protein kinase 7 (CDK7) plays key activity do not vary throughout the cell cycle. For several roles in the regulation of the eukaryotic cell cycle and CDKs, the phosphatases PP2C (Cheng et al., 2000) and in the control of transcription. These dual roles are in the kinase-associated phosphatase (KAP) (Poon and turn regulated by the phosphorylation state of the en- Hunter, 1995) have been identified that recognize zyme and the association with regulatory subunits. Like and dephosphorylate the phospho site on the activation most other CDKs, CDK7 requires association with an segment leading to inactive CDK. The crystal structure activatory cyclin, cyclin H, and phosphorylation on the of KAP in complex with phospho-CDK2 (pCDK2) (Song activation loop for activity. Assembly and activity are et al., 2001) showed that the major protein interface augmented by a third protein, the RING finger protein between the two proteins is formed by the C-terminal MAT1 (Devault et al., 1995; Fisher et al., 1995; Tassan helix of KAP and the C-terminal lobe of CDK2, regions et al., 1995). CDK7 CDK activation (CAK) activity is es- that are remote from both the KAP catalytic site and sential for cell cycle progression in vivo (Harper and the activation segment carrying the pThr160 residue of Elledge, 1998; Larochelle et al., 1998; Wallenfang and CDK2. At the KAP catalytic site, the CDK2 activation Seydoux, 2002). segment interacted almost entirely through the phospho CDK7/cyclin H or CDK7/cyclin H/MAT1 will phosphor- group and the structure required a movement of the ylate CDK1, CDK2, CDK4, CDK5, and CDK6 (Kaldis et activation segment in which it became unfolded and al., 1998; Rosales et al., 2003; Solomon et al., 1992) on drawn away from the kinase. Sequence comparisons their respective threonines in the activation segment showed that the KAP remote recognition site on CDK2 leading to activation of these kinases. Levels of CDK7 was not conserved in CDK7 and suggested that CDK7 in cells are low (Fisher and Morgan, 1994) and CDK7 would not be a substrate for KAP. location is predominantly nuclear (Tassan et al., 1994). As a first step toward understanding the recognition The in vivo activating kinase for human CDK7 phosphor- properties of CDK7, we report the crystal structure of CDK7 phosphorylated on Thr170 and in complex with *Correspondence: [email protected] ATP. The kinase is in an inactive conformation similar Structure 2068

Table 1. Summary of CDK7 Data Collection and Refinement Statistics 3.3 A˚ , ID29 3.0 A˚ , ID13 Combined Data

Space group and unit cell (A˚ )P21:aϭ 66.33, b ϭ 193.18, P21:aϭ 65.50, b ϭ 191.63, c ϭ 77.42, ␤ϭ95.12Њ c ϭ 75.79, ␤ϭ94.40Њ Resolution range (A˚ ) (last shell) 23.3–3.3 (3.48–3.3) 30.0–3.0 (3.16–3.0) 30.0–3.0 (3.16–3.0)

Rsym 0.105 (0.225) 0.165 (0.493) 0.143 (0.569) Observations 90970 (12322) 54240 (7970) 147115 (8046) Unique observations 28078 (4102) 29716 (4471) 36804 (4531) Mean(I)/␴(I) 10.4 (4.8) 6.1 (1.5) 8.5 (1.2) Completeness 96.4 (96.5) 80.0 (82.3) 94.9 (80.3) Multiplicity 3.2 (3.0) 1.8 (1.8) 4.0 (1.8) Refinement statistics

Rfactor 0.219

Rfree 0.296 Rmsd bond lengths (A˚ ) 0.017 Rmsd bond angles (Њ) 1.970 to that of inactive CDK2 but the activation segments of The results from CDK7 mass spectrometry show two the two CDKs differ. We describe the similarities and peaks with molecular mass of 39,217 and 39,141 corre- differences between CDK7 and CDK2 and other protein sponding to approximately 30% bisphosphorylated kinases with respect to their ATP binding sites and their CDK7 (39,061 ϩ 160) and 70% monophosphorylated protein recognition sites, including that for KAP. We CDK7 (39,061 ϩ 80). The results were identical for two correlate the structural observations with kinase activity separate preparations. In the crystal structure, we find measurements. only the monophosphorylated form. Thr170 is phosphorylated and Ser164 is not phosphorylated (Figure 1B). Results The pThr170 makes one hydrogen bond through its phosphate group to the main chain nitrogen of Gln22 The Structure of CDK7 from the glycine loop between ␤1/␤2 and is otherwise The structure of CDK7 was solved by molecular replace- exposed to the solvent (Figure 1C). The phospho-threo- ment using the structure of inactive CDK2 as a search nine approaches the ␥ phosphate of ATP (the distance object (Table 1). The CDK7 structure exhibits a typical between the ␥ phosphorous of ATP and the phospho- kinase fold comprising the N-terminal lobe (residues threonine phosphorus atoms is 6.7 A˚ ), but the side chain 13–96), with mostly ␤ sheet and one ␣ helix (␣C), and a of Gln22 partly intervenes between the two phosphates. C-terminal lobe (residues 97–311) comprised mostly of The structure suggests that autophosphorylation on ␣ helices (Figure 1A). The fold can be traced from residue Thr170 is unlikely in agreement with results in solution 13 to 311 in the 3.0 A˚ resolution electron density, whose (Martinez et al., 1997; Poon et al., 1994). Ser164 is ex- quality is enhanced by the 4-fold noncrystallographic posed on the surface of the protein. There is no addi- symmetry averaging. There is one break between resi- tional density surrounding this side chain to indicate dues 44–55, corresponding to the link between the ␤3 phosphorylation (Figure 1B). Ser164 is contained in a strand and the ␣C helix. The N-terminal residues 1–12 consensus sequence for CDK2 phosphorylation (SPNR and the C-terminal residues 312–346, that contain a pu- in single letter code) but has not been 100% phosphory- tative nuclear localization sequence, are not located in lated in insect cells. the electron density. ATP is bound between the two lobes and was clearly identifiable in all four copies of Comparison of CDK7 and CDK2 Structures the CDK7 structure. The conformation of CDK7 is similar The structures of CDK7 and CDK2 are similar with an to the inactive conformation of CDK2 in the absence rms deviation in C␣ positions for 251 residues of 1.25 A˚ . of cyclin A (De Bondt et al., 1993). The C-helix, which CDK7 has 44% sequence identity to CDK2 (Figure 2). contains the sequence NRTALRE (residues 56–62) in Major changes between CDK7 and CDK2 occur in three CDK7 corresponding to the PSTAIRE sequence motif in regions: (i) the activation segment (CDK7 residues 155– CDK2, is rotated about its axis and shifted toward the 174); (ii) the region where three parts of chain pack exterior of the protein compared with its conformation together comprising ␣D (CDK7 residues 101–112), the in the active CDK2/cyclin A complex (Jeffrey et al., 1995; ␤7/␤8 loop (CDK7 residues 146–149), and the C-terminal Russo et al., 1996a). All side chains in the region 56–62 region (CDK7 residues 297–311); and (iii) the region of (NRTALRE) are exposed. As a result, the interaction be- the L14 loop (residues 247-251) which is part of the KAP tween the glutamate from the C helix (Glu62) and a lysine recognition site and Cks1 regulatory protein binding site (Lys41), which stabilizes the lysine residue for interac- in CDK2 (Figure 3A). Changes in these regions have tion with the triphosphate of ATP in active protein ki- possible functional significance. In addition there are nases, is not made. The activation segment (Johnson also smaller changes at the N-terminal region and in the et al., 1996) is well localized in the electron density but loop regions between ␤1/␤2, ␤2/␤3 and ␤4/␤5 (Fig- has higher B factors than the rest of the molecule (aver- ure 3A). age B factors for main chain atoms for chain A: 42 A˚ 2; The activation segment of CDK7 adopts a different average main chain B factors for activation segment conformation to that observed for CDK2 and phospho- residues 155-182 in chain A: 58 A˚ 2). CDK2 (pCDK2) (Figure 3B). In pCDK2 (CDK2 phosphory- Crystal Structure of Human CDK7 2069

Figure 1. The Structure of CDK7/ATP Com- plex (A) A schematic diagram of CDK7 with secondary structural elements labeled. ATP is bound between the N- and C-terminal lobes. There is a break in electron density between the end of ␤3 and the start of ␣C. The activation segment is in a darker color. (B) The activation segment is phosphorylated on Thr170 and not on Ser164. The final Sig-

maA weighted 2Fo Ϫ Fc electron density map contoured at 0.94␴ for residues 164–172. (C) The activation segment of CDK7 in residues 163–171 showing the contacts of the pThr170 with the main chain nitrogen of Gln22 from the glycine loop between ␤1/␤2. This diagram and other figures were produced with AESOP (M.E.M. Noble, personal commu- nication).

lated on T160 but without the association of cyclin A) phosphorylation, the activation segment of CDK2 under- (Brown et al., 1999b) the structure is similar to inactive goes a dramatic change in conformation (Figure 3B). nonphospho CDK2 with the only difference that the acti- The separation of C␣ pThr170 (CDK7) to C␣ pThr160 in vation segment is disordered. In CDK7, at the start of pCDK2/cyclin A is 23.7 A˚ . the activation segment, the side chain of aspartate The second major change is at the C terminus where Asp155 of the DFG motif is turned away from the cata- residues 297–309 in CDK7 differ from the corresponding lytic site forming a hydrogen bond with Lys41, in a similar residues in CDK2. The CDK7 proline-rich C-terminal re- conformation to that observed for inactive CDK2. In this gion makes more intimate contacts with the rest of the position the aspartate is unable to participate in con- protein than with the corresponding region in CDK2. The tacts to the magnesium ion that interacts with the ␣ and interactions involve the end of ␣D and the ␣D/␣E loop ␤ phosphates of ATP in the activated form of CDKs. (residues 101–112, a region where there is little similarity CDK7 and inactive CDK2 activation segments differ from in sequence between CDK7 and CDK2), and the region this point (Asp155 in CDK7, Asp145 in CDK2) until they of the ␤7/␤8 loop residues146–149 (Figure 3A). The come back into register at residue Arg176 in CDK7 C-terminal region threads between these two loops and (Leu166 in CDK2) corresponding to the middle of the appears to displace the ␤7/␤8 loop. The end of the hinge P ϩ 1 loop. As in CDK2, there is a short two-turn helix region at ␤6 is also displaced and the ␣D helix in CDK7 (residues 158–165), which docks against the C helix lacks the final turn as seen in CDK2. The shifts allow keeping the latter in the inactive conformation through favorable contacts: for example, Pro308 interacts with interactions such as the packing of Phe156 against Val108 (␣D/␣E loop), Arg309 ion pairs with Glu147 (␤7/␤8 Val65. The activation segment takes a more open route loop), and Pro310 near the C terminus interacts with in CDK7 than in CDK2 (Figure 3B), resulting in the dis- Val100 and Ile101 (␣D helix). tance of 3.8 A˚ between C␣ atoms of pThr170 (CDK7) Protein kinases contain several protein interacting and Thr160 (CDK2). On association with cyclin A and sites on their surfaces. The MAP kinase family members Structure 2070

Figure 2. Sequence Alignment of CDK7 and CDK2 Showing the Assignment of Secondary Structural Elements in CDK7 Beta strands are shown as arrows and ␣ helices as shaded boxes. Short stretches of ␣

helix or 310 helix are shown as open boxes.

interact specifically with docking site sequences in sub- 5A). The differences arise from the overall differences strates (Sharrocks et al., 2000). These interactions may in protein conformation, partly promoted by the differ- confer pathway specificity and substrate selection and ence in conformation of the activation segments, and provide an additional recognition site that is separate the lack of magnesium ions in the CDK7 structure. from the catalytic substrate recognition site. The struc- Overall, the number of van der Waals contacts made ture of p38 in complex with docking peptides from two between ATP and CDK7 and between ATP and the inac- such interacting proteins, one from a transcription factor tive form of CDK2 are similar but the residues involved MEF2A and the other from an activating kinase MKK3b are different. ATP in CDK7 makes more extensive con- (Chang et al., 2002), show these peptides interacting at a tacts to the glycine loop than in CDK2 but fewer contacts site that is equivalent to that occupied by the C-terminal to the catalytic residues and the metal binding residues. ␤ ␤ region of CDK7 (i.e., the pocket between 7/ 8 loop and The adenine of ATP makes two hydrogen bonds to the ␣ ␣ the D/ E loop [Figure 3C]). In particular in CDK7 proline hinge region between the two lobes, as observed in residues 308 and 310 occupy the equivalent positions other kinase structures, and also contacts the hinge to the nonpolar residues in the sequence φa-X-φb that region residue Met94 (Figure 5B). The ribose in CDK7 is the hallmark of these p38-docking peptides. Although is closer toward the glycine loop than in CDK2, while the C-terminal region of CDK2 also occupies this pocket, the triphosphate moieties are very different especially the nonpolar interactions are not manifest to such a for the ␥ phosphates. In CDK7 two of the ␥ phosphate dramatic extent as in CDK7. oxygens hydrogen bond to main chain nitrogens of The third region of major difference is in the CDK Phe23 and Ala24 at the ␤1/␤2 turn making a well-local- insert region L14 where there is a deletion of one residue ized arrangement with the glycine loop. A third ␥ phos- in CDK7 with respect to CDK2 (Figures 2 and 3A). This phate oxygen hydrogen bonds to Lys41. The difference region forms part of the recognition site of pCDK2 for binding KAP (Song et al., 2001) (Figure 4A). In the in conformation of the activation segment between pCDK2/KAP complex, Lys237 of CDK2 makes an ion CDK7 and CDK2 allows the side chain of Ser161 to ␥ pair with Glu191 of KAP (Figure 4B). In CDK7, the corre- contribute a hydrogen bond to the phosphate in CDK7. sponding residue to Lys237 is Val247 (Figure 4C). The This residue occupies the position of Tyr15 in CDK2 small changes in sequence and conformation of this reflecting differences in the activation segments and region suggest that KAP would not be able to recognize glycine loops. In CDK7 Phe162, near the start of the pCDK7 and would not be able to dephosphorylate activation segment, stacks against Phe23, the residue CDK7. This hypothesis is further explored below. corresponding to Tyr15 in CDK2. The absence of metal ion in the CDK7 structure appears to have encouraged The ATP Binding Site the ␥ phosphate to seek hydrogen bonds with the gly- ATP bound to CDK7 has a slightly different conformation cine-loop main chain nitrogens rather than with the cata- and contacts than those of ATP bound to CDK2 (Figure lytic residues. Crystal Structure of Human CDK7 2071

Figure 3. Comparison of the Structures of CDK7 and CDK2 (A) A schematic diagram of CDK7 (in brown) and CDK2 (in cyan). There are significant changes in three major regions: the activation segment; the C-terminal region and contacts to the ␤7/␤8 loop and the end of the ␣D helix; and the L14 loop, the site of interaction of KAP in CDK2. (B) Comparison of the activation segments from CDK7 (brown), CDK2 (cyan), and activated pCDK2/cyclin A (green). The region from 155 (CDK7) DFG to 182 (CDK7) APE is shown. (C) The C-terminal region of CDK7 (brown) with the p38 structure (green) (PDB ID 1LEW) superimposed. The binding of the MEF2A docking peptide to p38 is shown in magenta. This overlaps the C-terminal region of CDK7.

-Figure 6A, lane 2, and Figure 6B) in agree) 2.5ف Kinase Assays and Substrate Specificity factor In order to determine the activation state of the crystal- ment with (Watanabe et al., 2000). The trimeric CDK7/ line CDK7 and to probe the influence of cyclin H and cyclin H/MAT228 complex is more active against the MAT1 on activity and specificity, we compared the rela- CTD substrate than against CDK2 (Figure 6A, lane 3, tive activities of CDK7, CDK7/cyclin H, and CDK7/cyclin and Figure 6B). Under our conditions, CTD phosphoryla- times over that observed for 4ف H/MAT228 against CDK2 and CTD as substrates. tion is increased by MAT228 (residues 228–309) is the minimal region of CDK7/cyclin H while the CDK2 CAK activity is not signifi- MAT1 that is able to stimulate CDK7/cyclin H activity cantly affected by the presence of MAT228. (Busso et al., 2000). In the presence of ATP, both CDK7/cyclin H and CDK7 has no activity against CDK2 but does have a CDK7/cyclin H/MAT228 will autophosphorylate cyclin H weak basal activity against the CTD (Figure 6A, lane 1 (unpublished data). Phosphorylation of cyclin H results and Figure 6B). CDK7/cyclin H complex is active with in a reduction in the activity of the dimeric CDK7/cyclin greater activity against CDK2 than against CTD by a H complex against both CDK2 and CTD (Figure 6C) Structure 2072

Figure 4. Comparison of the KAP Binding Site between pCDK2 and pCDK7 (A) Overall schematic diagram with CDK7 (brown) superimposed on the pCDK2/KAP complex (pCDK2 is in cyan and KAP in blue). (B) Details of the pCDK2/KAP binding site. pCDK2 is in cyan and KAP in magenta. There is an ionic interaction between Lys237 (pCDK2) and Glu191 (KAP). (C) Details of the changes at this site in CDK7 (in gold) with the key KAP residues superimposed. Val247 (which replaces Lys237 of CDK2) cannot interact favorably with Glu191. For further details see text. but no change in activity of the trimeric CDK7/cyclin CDK7/cyclin H or CDK7/cyclin H/MAT228 complexes H/MAT228 complex against either CDK2 or CTD (Figure produced by coinfection in insect cells, were found by 6C). It is possible that cyclin H phosphorylation destabi- mass spectrometry to have CDK7 60% bisphosphory- lizes the dimeric complex (thus reducing its activity), but lated and 40% monophosphorylated (data not shown). the presence of MAT1 provides additional stability in To analyze the influence of CDK7 phosphorylation state the trimeric complex and thus is able to counteract the on activity, we compared the CDK2 CAK activities before effects of phosphorylation of cyclin H. Thus, MAT1 may and after incubation with pThr160-CDK2/cyclin A and have an additional role in regulating CDK7 activity by ATP (Figure 6D). The CDK2 reaction appears to go to allowing the activity to escape regulation by cyclin H completion because subsequent incubations of these phosphorylation. preparations with pCDK2/cyclin A and ATP led to no Crystal Structure of Human CDK7 2073

CDK7/cyclin H and CDK7/cyclin H/MAT228 complexes and 2.8ف Figure 6D). There is an increase in activity of) /times for the additionally phosphorylated CDK7 3.7ف cyclin H and CDK7/cyclin H/MAT228 complexes, respectively. The lower activity of pCDK7/cyclin H with respect to pCDK7/cyclin H/MAT228 can most likely be ascribed to the autophosphorylation on cyclin H (described above) that causes a 35% reduction in activity of CDK7/cyclin H but no reduction for CDK7/cyclin H/MAT228 (Figure 6C). Activity against the CTD could not be tested under these conditions because pThr160- CDK2/cyclin A also phosphorylates CTD (Palancade and Bensaude, 2003).

KAP Dephosphorylates CDK2 but Not CDK7 The structure of the complex between KAP and pCDK2 (Song et al., 2001) showed that recognition at the catalytic site was achieved solely through the phosphate group but that the major determinant of specificity was located about 20 A˚ from the catalytic site where the C-terminal helix of KAP interacted with residues from the ␣G helix and the L14 loop of CDK2. The L14 region differs in CDK7 (Figure 4) resulting from sequence changes that involve the sequence DYV(247)-T in CDK7 (where – denotes a gap) and DYK(237)PS in CDK2 (Fig- ure 2). In order to test if these features destroy the docking site and abolish KAP activity, we tested KAP activity against CDK7 and constructed KAP-interacting mutants (KM) for CDK2 and CDK7. KM-CDK2 has the sequence Val237- in place of Lys237Pro238; KM-CDK7 has the sequence Lys247Pro in place of Val247-. These mutations create two chimeras: CDK2 with CDK7-L14 loop and CDK7 with CDK2-L14 loop. The profile of KAP activity against pThr160-CDK2, KM-pCDK2, CDK7, and KM-CDK7 is showed in Figure 7A. As expected, KAP is active against pCDK2 and inactive against CDK7. Dephosphorylation of pCDK2 results in a band shift in which dephosphorylated CDK2 mi- grates slower than the phosphorylated isoform (Figure 7A, lanes 2 and 1, respectively). No bandshifts can be Figure 5. Comparison of pCDK7/ATP and CDK2/ATP Interactions detected after KAP treatment of CDK7 either for the (A) Comparison of ATP bound to CDK7 (carbon atoms in green) and monophosphorylated (upper band) or bisphosphory- ATP bound to CDK2 (carbon atoms in yellow). lated (lower band) forms of CDK7 (Figure 7A, lanes 5 (B) Details of the interactions between CDK7 and ATP. and 6). The L14 loop mutations in KM-pCDK2 abolish (C) Details of the interactions between CDK2 and ATP. For further KAP activity (no bandshift detectable: Figure 7A, lanes details see text. 3 and 4). This result highlights the importance of the docking site for the regulation of KAP activity on pCDK2. further incorporation of phosphate and the phospho- However, KAP is not active against KM-CDK7 (Figure CDK7 ran as a single band under gel electrophoresis in 7A, lanes 7 and 8). Evidently the generation of a correct contrast to the two bands seen when both bis- and KAP docking site on CDK7 was not sufficient to make mono-phospho forms were present. After preincubation it a substrate. This may be because other effects are with pThr160-CDK2/cyclin A CDK7 showed weak activ- also important. For example, the rigidity of the pCDK7 ity, quantifiable as approximately 2% of the CDK7/cyclin activation segment compared with the disordered acti- H activity. Martinez et al. (1997) found that Xenopus vation segment in pCDK2 may make it less amenable CDK7 and CDK7/cyclin H can be activated by CDK2/ for conformational change to reach the KAP catalytic cyclin A or CDK1/cyclin B in reticulocyte-translated ly- site. We note that the relative intensity of the bands in sates with pCDK7 achieving approximately one-third of Figure 7, show that KM-CDK7 is approximately 70% the activity of the CDK7/cyclin H complex. From our bisphosphorylated and 30% monophosphorylated (Fig- results, we conclude that in human phospho-CDK7, in ure 7A, lane 7), while CDK7 is 30% bisphosphorylated contrast to Xenopus CDK7, does not exhibit appreciable and 70% monophosphorylated (Figure 7A, lane 5). CDK2 CAK activity. In order to verify that these mutations did not affect The effects of phosphorylation are more evident in the the overall folding and the other binding sites of CDK2 Structure 2074

Figure 6. Kinase Activities of CDK7, CDK7/ Cyclin H, and CDK7/Cyclin H/MAT228 (A) Autoradiograph of CDK2 and CTD phosphorylated by CDK7 (lane 1), CDK7/cyclin H (lane 2), and CDK7/cyclin H/MAT228 (lane 3). The reactions were performed as described in Experimental Procedures. (B) Relative CDK2 and CTD phosphorylation activities for CDK7 CDK7/cyclin H and CDK7/ cyclin H/MAT228 from results shown in (A). Cyclin H in the dimeric complex generates CAK activity on CDK2 making CDK2 the pre- ferred substrate for CDK7. MAT228 in the trimeric complex increases CTD phosphorylation without affecting CAK activity. (C) Autophosphorylation on cyclin H de- creases CDK7/cyclin H activities on CDK2 and CTD without affecting CDK7/cyclin H/ MAT228 activities. (D) Effect of CDK7 phosphorylation by pThr160-CDK2/cyclin A on CAK activity. Min- imal basal activity is induced in CDK7 (change too small to be observed in this figure). CDK7/ cyclin H and CDK7/cyclin H/MAT228 com- 2.8ف plexes showed an increase in activity of times after preincubation with 3.7ف and pThr160-CDK2/cyclin A. and CDK7, we compared activity of wild-type and mu- crystallization showed no activity against CDK2 but did tant proteins (Figure 7B). Phosphorylation of CDK7 by exhibit basal activity against the CTD from RNA polymer- pThr160CDK2 or KM-pThr160CDK2 are similar (Figure ase. The lack of conventional activity against CDK2 is 7B, lanes 1 and 2) and the activatory effects of cyclin A consistent with the inactive conformation of the kinase on pCDK2 or KM-pCDK2 for phosphorylation of CDK7 observed in the crystal structure. Further phosphoryla- are also similar (Figure 7B, lanes 3 and 4). These results tion of CDK7 by pCDK2/cyclin A in order to achieve full show that KM-pCDK2 has been phosphorylated by Civ-1 phosphorylation on both Thr170 and Ser164, led to no during expression to give the active enzyme and that significant CAK activity against CDK2. Phosphorylation mutation of pCDK2 L14 loop does not affect interactions of the activation segment in CDK7 is insufficient to trig- with either cyclin A or with the substrate CDK7. Both ger the conformational changes that convert an inactive CDK7 and KM-CDK7 are able to phosphorylate CTD kinase to an active kinase. (Figure 7B, lanes 5 and 6). The higher activity of KM- Association of CDK7 with cyclin H and with cyclin CDK7 is ascribed to its different phosphorylation state H/MAT228 leads to significant activity against both in respect to CDK7 (described above). We conclude CDK2 and the CTD. In these complexes there is a higher that mutation of CDK7 L14 loop does not affect activity amount of bisphosphorylated CDK7 than monophosph- against the CTD. orylated CDK7 (ratio 60:40) produced by the insect cells. We find that the CDK7/cyclin H complex has a preference for CDK2 over CTD of about 2.5-fold at substrate Discussion concentrations of 7.6 ␮M, as others have also observed (Watanabe et al., 2000) but that with the CDK7/cyclin The crystal structure of CDK7 shows that the overall H/MAT228 complex the substrate preference is shifted fold of the kinase is similar to that of inactive CDK2, as by a ratio of about 1.4 in favor of the CTD. Others have expected from their sequence similarity. Major differ- observed a more dramatic shift in substrate specificity ences occur in the activation segment, the C-terminal on association with MAT1 (Larochelle et al., 1998; Yan- region and the L14 loop, which is part of the CDK2 kulov and Bentley, 1997). The MAT1 construct of resi- recognition site for KAP. Mass spectrometry indicated dues 228-309, which was used in our experiments, has a 30:70 ratio of bisphosphorylated and monophosphory- been identified as the minimal construct need to provide lated forms of CDK7 produced by expression from bacu- activation of CDK7/cyclin H (Busso et al., 2000). How- loviral vectors in insect cells but the crystal structure ever, the N-terminal RING finger domain is important for contains only the monophosphorylated form in which transcription activation and promotion of phosphoryla- Thr170 is phosphorylated. Evidently crystallization has tion of the CTD while the middle region comprising selected the monophosphorylated form from the mix- a coiled-coil allows binding of CDK7/cyclin H/MAT1 ture. In contrast to pThr160CDK2 where the phosphory- through interactions with both XPD and XPB helicases lated activation segment is disordered, CDK7 shows an (Busso et al., 2000). In contrast to CDK7 alone, further ordered activation segment. The conformation of the phosphorylation of CDK7/cyclin H and CDK7/cyclin activation segment is different from that of CDK2 and H/MAT228 by pCDK2/cyclin A led to an enhancement from that of pCDK2/cyclin A but is closer to that of of activity against CDK2 substrate of 3.7-fold, sug- the inactive CDK2. The CDK7 preparation prepared for gesting that phosphorylation on both Thr170 and Ser164 Crystal Structure of Human CDK7 2075

al., 2001) explained why KAP will only dephosphorylate pCDK2 when it is in its folded state and not when it is denatured. There are changes in sequence in the KAP recognition site of CDK2 in CDK7, which suggested that this recognition site would be disrupted in CDK7. This has been confirmed by the present work, which has shown that CDK2 is a substrate for KAP but not CDK7. We used site directed mutagenesis to change the L14 loop of CDK2 to resemble that of CDK7 (resulting in KM- CDK2). The changes in sequence of just two amino acids were sufficient to disrupt the recognition site and KAP showed no activity against the KM-CDK2. In contrast engineering the CDK2 sequence into the CDK7 L14 loop (KM-CDK7) did not give a protein substrate for KAP. We conclude that other factors, such as the greater rigidity of the phospho-activation segment, could also contribute to the resistance of CDK7 to dephosphorylation by KAP. Protein kinases are major targets for drugs. There are several compounds that are either on the market or in advanced clinical trials for treatment of cancer, diabetes and inflammatory diseases (Noble et al., 2004). One of the major problems is to develop a compound that is selective for a particular kinase or signaling pathway. Almost all drugs developed so far target the ATP binding site. We compared the ATP site in two homologous kinases CDK2 and CDK7 to see if there are features that might confer selectivity. The major differences between Figure 7. KAP Activity against CDK2, KM-CDK2, CDK7, and KM- CDK7 and CDK2 ATP sites occur at the “CDK2 Lys89 CDK7 and Kinase Assays pocket” or “hydrophobic pocket” (Fabbro et al., 2002). (A) KAP activity. Dephosphorylation of CDK2 is detectable by band- This pocket has been targeted successfully by CDK2 shift (lanes 1 and 2) but not for KM-CDK2 (lanes 3 and 4); no changes inhibitors (Davies et al., 2002; Davis et al., 2001; Gray are detectable in the relative intensity of monophosphorylated and et al., 1998) to provide potent and selective inhibitors. diphosphorylated CDK7 (lanes 5 and 6) or for KM-CDK7 (lanes 7 In CDK7 the residue corresponding to Lys89 at the bot- and 8). The relative shift of bands between CDK7 and KM-CDK7 arise from the difference in phosphorylation state of CDK7 (discussed in tom of the pocket is Val100 (Figures 5B and 5C). This text). change together with other changes in the region such (B) Comparison of kinase activities of CDK2 and CDK7 and their as Thr96 (in place of Gln85 in CDK2), and the presence respective L14-loop mutants. CDK7 was used as substrate for of Pro310 creates a pocket of greater nonpolar character CDK2; CTD was used as substrate for CDK7. (See text for further in CDK7 than in CDK2. In CDK4, Lys89 of CDK2 is re- details.) placed by a threonine and this change has been ex- ploited in the development of specific CDK4 inhibitors are required for maximal activity. The change in sub- (Beattie et al., 2003; Breault et al., 2003; Honma et al., strate specificity conferred by MAT1, which shifts the 2001a, 2001b; Soni et al., 2001) and a CDK4-like mutant preference to the CTD over CDK2, raises intriguing ques- of CDK2 (Ikuta et al., 2001). In CDK7 this hydrophobic tions that can only be answered by structural studies on pocket offers the possibility for designing selective in- the dimeric and trimeric complexes and their association hibitors. with the substrates. The structure of CDK7 joins the structures of CDK2 Protein/protein interactions at sites remote from the (Brown et al., 1999a; De Bondt et al., 1993; Jeffrey et catalytic site play key roles in many processes of protein al., 2001; Russo et al., 1996a), CDK5 (Tarricone et al., kinase substrate recognition (e.g., MAPK family; CDK2; 2001), and CDK6 (Brotherton et al., 1998; Russo et al., GSK; PLK1). The positions of these sites have been 1998; Schulze-Gahmen and Kim, 2002) as representa- elucidated in a number of structural studies with pep- tive structures of the CDK family. Each of these exhibits tides from the cognate substrate proteins (Bax et al., different regulatory and substrate specificity properties. 2001; Chang et al., 2002; Cheng et al., 2003; Dajani et For CDK7 further work is required to understand the al., 2003; Elia et al., 2003; Lowe et al., 2002; Russo et activation by regulatory domains and to understand why al., 1996b) and demonstrate that different parts of the CDK2 can phosphorylate CDK7 and CDK7 can phos- kinase surface or regulatory domains may be involved phorylate CDK2 but neither can autophosphorylate. The in such recognition. These studies have not yet shown role of additional protein recognition sites in these pro- how the docking sites and the catalytic sites communi- cesses should provide a fascinating explanation of sub- cate, if at all. The crystal structure of KAP in complex strate specificity as will understanding other processes with pCDK2 represents one of the few examples where such as the interactions of cyclin H with the U1 snRNA an enzyme in complex with its complete phospho-pro- that provide a link between transcription and RNA protein substrate is known. The structural results (Song et cessing (Kwek et al., 2002) and the interactions of CDK7 Structure 2076

with the Hepatitis C virus core protein that could link Kinase Assays viral infection to cell cycle regulation (Ohkawa et al., CDK7, CDK7/cyclin H, and CDK7/cyclin H/MAT228 activities were 2004). measured by following the incorporation of radiolabeled phosphate into substrate. CDK2 (2.6 ␮g ϭ 76 pmol) or GST-CTD (5 ␮g ϭ 76pmol) as substrates were incubated with 100 ng of CDK7, CDK7/cyclin H, or CDK7/cyclin H/MAT228 in 10 ␮l kinase buffer (0.1 mM ATP, 10 mM Experimental Procedures 32 MgCl2, 50 mM Tris-HCl (pH 7.5), 1 ␮Ci ␥- P-labeled ATP (Amersham Biosciences). The reaction mixtures were incubated for 5 min at All reagents, unless specified otherwise, were purchased from 20ЊC and terminated by the addition of SDS sample buffer. Auto- Sigma-Aldrich (St. Louis, MO). phosphorylated CDK7/cyclin H and CDK7/cyclin H/MAT228 were obtained by incubating the each complex in 50 mM Tris-HCl (pH Њ Expression, Purification, and Mass Spectrometry 7.5), 0.1 mM ATP, and 10 mM MgCl2 for 30 min at 20 C. Fully phos- pBS-CDK7 vector, kindly provided by Prof. David Morgan (Dept. of phorylated CDK7, CDK7/cyclin H, and CDK7/cyclin H/MAT228 were Physiology, University of California, San Francisco, CA) and Prof. obtained incubating each with 50 ng of pThr160-Cdk2/cyclin A in Robert P. Fisher (Memorial Sloan Kettering Cancer Center, New 50 mM Tris-HCl (pH 7.5), 0.1 mM ATP, and 10 mM MgCl2 for 30 Њ York), was used as a template for the subsequent PCR. CDK7 was min at 20 C. Samples were analyzed on 12% SDS–PAGE gels and cloned into the vector pVL1392 (Pharmingen BD, Franklin Lakes, exposed to the PhosphorImager image plate (Molecular Dynamics, NJ) modified with the insertion of GST and PreScission protease Amersham Biosciences). Pixels were quantified using the ImageJ ϭ cleavage site. CDK7-expressing baculovirus was obtained by software (NIH) and converted into MBq using the equation: pixels ϩ transfecting 2 ϫ 106 Sf21 cells with 2 ␮g of plasmid DNA and 0.5 A ln(Mbq) B. A and B are experimental parameters calculated ␮g of linearized baculovirus DNA for 5 days using BaculoGold Trans- from the image plate calibration. Data were normalized with respect fection kit (Pharmingen BD). After two rounds of amplification, to the enzyme concentration. expression was obtained by infecting Sf21 cells with GST-CDK7 baculovirus (MOI 0.3) at 27ЊC for 3 days. Cells were collected by KAP Assay centrifugation at 1000rpm and resuspended in 50 mM Tris-HCl (pH Dephosphorylation reactions were performed by incubating 0.5 ␮g 8), 250 mM NaCl, 10% glycerol, and 10 mM DTT and Complete of pThr160-CDK2 or KM-pThr160-CDK2 with 0.2 ␮g of KAP or 2 ␮g protease inhibitor cocktail (Roche, Basel, Switzerland). Cells were of CDK7 or KM-CDK7 with 0.8 ␮g of KAP in 50 mM Tris (pH 7.5), disrupted by sonication and the lysate, after clarification by centrifu- 150 mM NaCl, 2 mM DTT, and 0.5 mM EDTA for 1 hr at room gation at 10,000 rpm, was loaded on GSH-Sepharose (Amersham temperature. Samples were then loaded on SDS-PAGE gel and ana- Biosciences, San Francisco, CA). Resin was washed with 10 column lyzed for bandshift by Coomassie blue stain. volumes of the lysis buffer and then with 10 column volumes of 50 mM Tris-HCl (pH 8), 250 mM NaCl, 10% glycerol, 1 mM DTT, and Crystallization 1 mM EDTA. Cleavage was performed overnight with 1 column volume Sitting drop crystallization trials were performed at 4ЊC using Molec- of the above buffer containing 40 ␮l of PreScission (Amersham ular Dimensions (Soham, UK) Structure Screen I and II. Drops were Biosciences) protease/ml resin. Cleaved protein was recovered in set up using Genesis ProTeam 150 crystallization robot (Tecan, 2 column volumes of the same buffer and loaded onto Superdex Reading, UK) and CrystalQuick 96-well plates (Greiner bio-one, 200 26/60 (Amersham Biosciences). Size-exclusion chromatogra- Longwood, FL) mixing 0.2 ␮l of CDK7 (7 mg/ml) with 0.2 ␮l of testing phy was run using 50 mM Tris-HCl (pH 8), 200 mM NaCl, 10% solutions. Drops were equilibrated by vapor diffusion with 100 ␮lof glycerol, and 1 mM DTT. its testing solution in the plate reservoir. Final crystallization condi- Cyclin H baculovirus was a gift from Prof. David Morgan (Dept. tions were identified as 0.1 M Na Citrate (pH 6.4), 0.2 M Na Acetate of Physiology, University of California, San Francisco, CA). pGEX- (pH 6.4), 20% PEG 4000, 10% glycerol, 200 mM nondetergent sul- MAT1, provided by Prof. Erich Nigg (Max Planck Institut, Martinsried, phobetaine 201 (NDSB201), and 2 mM ATP. The solubilizing agent Germany), was used as a template for subsequent PCR. MAT228 NDSB201 helped prevent excessive nucleation and promoted crys- (corresponding to the C-terminal region of MAT1 residues 228–309) tal growth. The crystals were transferred to 0.1 M Na Citrate (pH was cloned into the vector pVL1392 (Pharmingen BD) modified with 6.4), 0.2 M Na Acetate (pH 6.4), 20% PEG 4000, 20% glycerol before the insertion of MBP and PreScission protease cleavage site. CDK7/ freezing in liquid nitrogen. cyclin H and CDK7/cyclin H/MAT228 complexes were produced by coinfection of respective viruses in Sf21 cells. Purifications were X-Ray Data Collection, Data Processing, carried out as described above. and Structure Solution ϫ 10 ϫ 5 ␮m). An initial dataset was 50ف) pGEX-CTD was provided by Dr. Andre Furger (Dept. of Biochemis- The crystals were small try, University of Oxford, UK) and used to transform DH5␣ cells. Cells collected at beamline ID29, ESRF to 3.3 A˚ (Table 1.). The crystals were induced overnight with 0.25 mM IPTG at 20ЊC and sonicated in were space group P21 with four molecules per asymmetric unit. A 50 mM Tris-HCl (pH 8), 200 mM NaCl, 10% glycerol, and 10 mM second data set was collected at the micro-focus beamline, ID13 DTT and Complete protease inhibitor cocktail (Roche). Clarified ly- (ESRF) using a focal spot size of 10 ␮m . The high brightness of the sate was loaded on GSH-Sepharose (Amerham Biosciences) and beam allowed data collection to 3 A˚ resolution (Table 1). However, resin was then washed with 10 column volumes of lysis buffer and severe radiation damage allowed only 5–10 degrees of data to be 10 column volumes of in 50 mM Tris-HCl (pH 8), 200 mM NaCl, 10% collected from one part of the crystal before it was necessary to glycerol, and 1 mM DTT. GST-CTD was then eluted with 2 column translate the crystal and continue data collection using an unex- volumes of 50 mM Tris-HCl (pH 8), 200 mM NaCl, 10% glycerol, and posed region. Each of these data segments was indexed separately 10 mM glutathione. with the program MOSFLM (Leslie, 1992). Only 80% overall com- CDK2, pThr160-CDK2, and pThr160-CDK2/cyclin A were pro- pleteness was achieved because of radiation damage. The 3 A˚ data duced as previously described (Brown et al., 1999a) set was scaled together with the 3.3 A˚ dataset previously recorded. pET28a-KAP expression vector was kindly provided by Prof. Da- The combined data set with addition of reflections in both the high vid Barford (Institute of Cancer Research, London, UK) and subse- and low resolution shells resulted in data that gave a successful quently used to purify KAP as described (Hanlon and Barford, 1998). structure solution. Mutagenesis of pThr160-CDK2 and CDK7 to produce KAP interac- The structure was solved by molecular replacement with the pro- tion mutants (KM) were performed using the Site Directed Mutagen- gram MOLREP (CCP4, 1994) using a modified model of inactive esis Kit (Stratagene, La Jolla, CA, USA) and following manufacturer’s CDK2 (Protein Data Bank accession code 1HCK) as the search instructions. These mutants were expressed and purified adopting object. MOLREP found three copies of the model (Rfactor ϭ 0.513, the same procedures used for wild-type proteins. CC ϭ 0.294). Examination of the resulting electron density maps Mass spectrometric analyses were performed on VG Fisons “Plat- revealed a further copy. A search in MOLREP using the previous form” single quadrupole mass spectrometer using samples desalted solution as fixed input and a search model corresponding to the by drop dialysis. C-terminal domain of CDK2 (residues 104–152, 164–237, and 240– Crystal Structure of Human CDK7 2077

298) successfully identified the orientation of the fourth molecule CCP4 (Collaborative Computational Project Number 4) (1994). The

(Rfactor ϭ 50.5, CC ϭ 0.306). The difficulty in locating the fourth mole- CCP4 suite: programs for protein crystallography. Acta Crystallogr. cule arose because of a clash between the second and fourth mole- D Biol. Crystallogr. 50, 760–763. cules in the asymmetric unit in the region following the hinge corre- Chang, C.I., Xu, B.E., Akella, R., Cobb, M.H., and Goldsmith, E.J. sponding to residues 97–100 of CDK2. This initially suggested that (2002). Crystal structures of MAP kinase p38 complexed to the dock- there were two different conformations of CDK7 present within the ing sites on its nuclear substrate MEF2A and activator MKK3b. Mol. asymmetric unit. However, upon rebuilding it became apparent that Cell 9, 1241–1249. CDK7 differed from CDK2 in this region and the same backbone Chen, J., Larochelle, S., Li, X., and Suter, B. (2003). Xpd/Ercc2 regu- conformation was in fact present in all four copies. As a result, 4-fold lates CAK activity and mitotic progression. Nature 424, 228–232. averaging was applied to maps during rebuilding. Rebuilding was carried out using O (Jones et al., 1991), followed by rounds of refine- Cheng, A., Kaldis, P., and Solomon, M.J. (2000). Dephosphorylation ment in REFMAC5 (Murshudov et al., 1997) resulting in a final struc- of human cyclin-dependent kinases by protein phosphatase type 2C␣ and ␤2 isoforms. J. Biol. Chem. 275, 34744–34749. ture with Rfactor ϭ 0.219, Rfree ϭ 0.296 (Table 1). Cheng, K.-Y., Lowe, E.D., Sinclair, J., Nigg, E.A., and Johnson, L.N. Acknowledgments (2003). The crystal structure of the human polo-like kinase polo- box domain and its complex with a phosphopeptide. EMBO J. 22, We wish to thank the scientists at ESRF for their help with data 5757–5768. collection, namely David Flot and Christian Riekel at ID13 and the Dahmus, M.E. (1996). Reversible phosphorylation of the C-terminal scientists at ID29. We are grateful to David Barford, Robert Fisher, domain of RNA polymerase II. J. Biol. 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Accession Numbers

The coordinates have been deposited in the Protein Data Bank (code 1UA2).