The structure of CDK9/CycT1 and comparison with CDK2/CycA. Schematic overall representations of CDK9/CycT1 (left; CDK9 is in green, CycT1 in brown) and CDK2/CycA (right; CDK2 is in orange, CycA in magenta). The phospho-threonine residues in the activation segments are marked by a red circle. The CDK structures have been aligned and the 26° rotation of CycT1 with respect to the position of CycA is indicated. This and other structure figures were prepared with PyMOL (WL DeLano, The PyMOL Molecular Graphics System; DeLano Scientific, San Carlos, CA, USA, 2002).

CDK9 structure. (A) The structures of CDK9 (green) and CDK2 (orange) superimposed. The cyclins have been omitted for clarity. (B) A close-up of pThr186 in CDK9 with the final 2F_o–F_c electron density and side chain contacts. CDK2 pThr160 and contacts (orange) are superimposed. (C) Sequence comparison of CDK9, CDK2 and other CDKs. Secondary structural elements are indicated for CDK9 and CDK2 in green and orange, respectively. Residues conserved in all CDKs are shown in green and those that are similar in yellow. Residues invariant in CDK9s from human, mouse, chicken, worm, fly and fish but not conserved in the other CDKs are boxed with a red frame in the CDK9 sequence (Supplementary Figure S7). Black circles below the sequences indicate CDK9 residues in contact with CycT1 (<3.5 Å). Red open circles indicate basic residues that contribute to the positively charged surface of CDK9 (Supplementary Figure S4). Black asterisks correspond to autophosphorylation sites.

AMPPNP and flavopiridol binding. (A) Details of the interaction of AMPPNP with CDK9/CycT. The CDK9 main chain is represented in green together with the side chains of residues within 3.5 Å of AMPPNP. (B) Details of the interaction of flavopiridol with CDK9/CycT1 and conformational changes. CDK9 in the flavopiridol complex is in green and in the AMPPNP complex in grey. (C) Details of the flavopiridol CDK9 interactions and a schematic representation of flavopiridol. The electron density maps are the final 2F_o–F_c maps contoured at 1σ around the ligand (A) AMPPNP and (B) flavopiridol.

Catalytic activity of CDK9/CycT1 on RNA Pol II CTD substrate. (A) CDK9-T186A/CycT1-298 showed no activity on CTD in contrast to CDK9-WT/CycT1-298 or the mutant CDK9-S347E/CycT1-298. (B) CDK9/CycT1-288 activity towards the CTD after preincubation with ATP and removal of excess ATP/ADP. (C) Autophosphorylation reactions for active and kinase-dead CDK9-D167N/CycT1-288 complexes. CDK9-D167N/CycT1-288 or CDK9/CycT1-288 variants as substrates (0.6 μg, (++)) were incubated with or without catalytic amounts (50 ng, (+)) of active CDK 9/CycT1-288.

Comparison of CycT1 and CycA. (A) Schematic diagrams of CycT1 (left) and CycA (right) with secondary structural elements labelled. The colours are the same as Figure 1 but with the H_N and H_C helices in green and red, respectively, and residues in contact with the CDK in yellow. The orientation of CycA is as in Figure 1. CycT1 has been aligned with CycA for comparison. (B) Structure-based sequence alignment of transcriptional and cell cycle cyclins. Residues conserved in all eight cyclins are in green and those that are similar in yellow. Cyclin A, B, E and T1 residues in contact with their cognate CDK (distance <3.5 Å) are shown highlighted in red. Secondary structure and numbering for CycT1 are shown above the sequences.

Details of CDK9/CycT1 interactions. (A) The contacts between CycT1 and the N-terminal residues of CDK9. The view, as indicated in the small overall figure, is 120° rotated about the horizontal axis with respect to Figure 1. Secondary structural elements, shown for guidance but not in contact, are in grey. The colour code is as in Figure 1. (B) The major contacts between CDK9 and CycT1 in the vicinity of the CDK9 αC helix. For further details see text. (C) Selected residues showing the substrate-binding site and phospho-threonine environment for CDK9/CycT1 (left) and CDK2/CycA (right). The substrate heptapeptide bound to CDK2/CycA (Brown et al, 1999) is shown in cyan.

Mass spectrometry analysis of CDK9-FL/CycT1-298 phosphorylation state. (A) CDK9, in the purified CDK9/CycT-298 complex, is ∼80% mono-phosphorylated. The expected MW value of the non-phosphorylated protein is 42800.5. The observed masses are: 0P, 42 803; 1P, 42 881. (B) Phosphorylation is homogeneous on Thr186. MS/MS spectrum of the m/z 665.27²⁺ ion corresponding to CDK9-phosphorylated peptide 179–188. A phosphate loss is observed (m/z 616.29²⁺). Peaks marked with an asterisk correspond to dehydroamino-2-butyric acid containing y″- and b-ions and indicate phosphate location on Thr186 (zoom panel). (C) Distribution of phosphorylation states for CDK9 following autophosphorylation reaction. The observed masses are: 1P, 42 876; 2P, 42 960; 3P, 43 038; 4P, 43 115; 5P, 43 201; 6P, 43 273; 7P, 43 433. (D) Autophosphorylation reaction does not affect CycT1-298 phosphorylation state. The expected MW value for CycT1 34 157 corresponds to the observed mass 34 157. (E) Analysis of autophosphorylated CDK9/CycT1-298 by LC/ESI-MS. Left: starting preparation as purified from insect cells. Right: same preparation following limited in vitro autophosphorylation reaction. The expected MW value of the non-phosphorylated protein is 42 800.5. The observed masses are: 0P, 42 797; 1P, 42 878 (left panel) and 0P, 42 801; 1P, 42 880; 2P, 42 960; 3P, 43 043 (right panel). (F) m/z region (1200–1340) of the MALDI mass spectra from tryptic digests of CDK9 showing the peaks corresponding to peptide 179–188 both in parent and in phosphorylated forms. (a) Digest of CDK9 starting preparation as purified from insect cells. (b) Digest of the same preparation following in vitro autophosphorylation reaction.