Crystal structure of human cyclin K, a positive regulator of cyclin-dependent kinase 9

Abstract

Cyclin K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, referred to collectively as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 A resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell-cycle inhibitor p27(Kip1). Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K structure as a template reveals that the two proteins have similar structures, as expected from their high level of sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9-cyclin K and CDK9-cyclin T1 complexes.

Figure 1

(a) Sequence comparison of human cyclin K (residues 11-267) and cyclin T1 (residues 1-272). The protein sequences were aligned using the program CLUSTALW. Hyphens represent gaps inserted for optimum alignment. Identical residues are shown in white on blue background and similar residues are highlighted in yellow. The secondary structure elements of cyclin K, assigned by the program STRIDE using both the hydrogen bonding and backbone torsion angles, are indicated at the top. Helical regions in the N- and C-terminal cyclin boxes are colored green and red, respectively. The cyclin T1-specifc motif TRM is boxed and residue C261 that is critical for binding to HIV-1 Tat is shown in red. (b) Stereo view of a weighted 2F_obs−F_calc electron density map calculated at 1.5 Å and contoured at 2.9 σ. Carbon, nitrogen, oxygen, and sulfur atoms are shown in magenta, blue, red, and yellow, respectively. The figure was made using BOBSCRIPT and POV-Ray (www.povray.org).

Figure 2

Structural and topological comparison of cyclins K, C, and H. Ribbon and topology diagrams of human cyclin K (a), S. pompe cyclin C (b), and human cyclin H (c). In the ribbon diagrams the corresponding helices in the three cyclins have identical colors. In the topology diagrams the helices are shown as cylinders, with the N-terminal cyclin box helices H1–H5 colored green and the C-terminal box helices H1′–H5′ colored red. The figure was made using PYMOL (www.pymol.org) and TOPDRAW.

Figure 3

(a) Model of the CDK9–cyclin K complex generated using the program MODELLER based on the CDK2–cyclin A structure (PDB code 1QMZ). The proteins are depicted as ribbon models with superimposed semitransparent molecular surfaces. CDK9 is colored blue and the PITALRE region is colored magenta. Cyclin K is colored as in Figure 2(a). (b) Amino acids at the CDK9–cyclin K interface. Side chains of residues ideally situated for binding are colored cyan (CDK9) and yellow (cyclin K). (c) Surface representation of the CDK2–cyclin A complex bound to p27^Kip1 (red ribbon) (PDB code 1JSU). (d) p27^Kip1 is shown in the modeled CDK9–cyclin K complex. Binding of the N-terminal p27^Kip1 region to cyclin K is obstructed by the C-terminal end of helix H4 (cyan) and helix H4a (green).

Figure 4

(a) Stereo view of a human cyclin T1 model (colored gray) superimposed to the cyclin K structure (colored orange). (b) and (c) Surface representation of the CDK9-interacting surfaces of cyclins K and T1, respectively. Identical, similar, and different cyclin residues at the interface with CDK9 are colored blue, yellow, and red, respectively. Figure (a) was made using PYMOL and Figures (b) and (c) using GRASP.