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I.
Introduction This exhibit displays molecules in the left part of the screen,
and text that addresses structure-function relationships of
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I.
Introduction
Progress in the cell cycle is driven by oscillations in the activities of cyclin-dependent kinases, or CDKs. CDKs are catalytically inactive until bound by a cognate, regulatory cyclin. Various cyclins are transiently produced during different cell cycle stages and partially activate their CDK partners. This activation is augmented by phosphorylation of a key CDK threonine residue by a CDK-activating kinase (CAK), promoting full kinase activity. For a short review of the cell cycle and the role of cyclins and their dependent kinases, see Figure 1. The loss of control of CDK activity is a hallmark of several diseases, including cancer (Malumbres, 2014).
CyclinA regulates several steps of the cell cycle by virtue of its ability to activate two different CDKs: CDK2 and CDK1 (Yam, et al., 2002; Bendris, et al., 2011). Association of nuclearly localized cyclinA with CDK2 is required for passage into the S phase of the cell cycle, with the cyclinA-CDK2 complex playing a role in the initiation of DNA replication (Coverley, et al., 2002). Additionally, cyclinA-CDK2 is involved in phosphorylating particular DNA replication proteins during the S phase, preventing the formation of post-initiation replication complexes and thus ensuring that replication is restricted to one round (Yam, et al., 2002; Coverley, et al., 2002; Woo, et al., 2003).
This exhibit, based on the work of Jefferey, et al. (1995) and Schulze-Gahmen, et al. (1996), will demonstrate how cyclinA activates CDK2, showing that binding causes large conformational changes that result in the formation of a functional CDK2 active site by movements of key structural elements including the PSTAIRE helix, the T-loop, and the N-terminal β-sheet (see below).
II. CyclinA and CDK2 Structure
CDK2 contains a β-sheet-rich amino terminal lobe and a carboxy terminal lobe with multiple α-helices. ATP is nestled in a deep cleft between these lobes, the site of the CDK2 active site (see below). CyclinA interacts with CDK2 on one side of the catalytic cleft, interfacing with both CDK2 lobes along a broad surface.
Note: in the 1FIN structure shown, cyclin is a partial fragment which nevertheless posesses full CDK2 binding and activation attributes and contains core sequences shared by all cyclins (Jeffrey, et al., 1995).
Essential to the cyclin-CDK interface is the PSTAIRE helix, containing the PSTAIRE sequence motif named for the one-letter designations of its amino acids: proline-serine-threonine-alanine-isoleucine-arginine-glutamate. This motif is conserved among cyclin-dependent kinase family members. The amino acid sequence in the PSTAIRE region has been shown to confer cyclin specificity to CDKs (Pines, 1994).
As will be seen below, the PSTAIRE helix rotates and moves into the CDK catalytic cleft in response to cyclin binding. Note the position of the PSTAIRE glutamate in the active site cleft of the kinase. Also interacting with cyclin is the T-loop of CDK2. This loop swings away from the catalytic cleft in response to cyclin binding (see below), making room for the PSTAIRE helix and opening access to the active site. This movement (see below) exposes a target threonine residue that is phosphorylated by CDK2-activating kinase (CAK), endowing CDK2 with full catalytic activity.
The CyclinA fragment shown at left contains 12 α-helices with two nearly identical structural repeats (folds) accounting for 10 of these. Fold1 and Fold2, despite sharing a common structure, are only 12% identical in sequence! The folds are joined by a 5 amino acid linker. A long α-helix upstream (N-terminal) to Fold1 packs against Fold2.
Fold1 represents most of the "cyclin box," an ~100 residue conserved sequence found in other cyclins. This fold plus the N-terminal helix of cyclinA are responsible for the majority of CDK2 binding, with elements of Fold1 contacting CDK2's N-terminal β-sheet, T-loop, and PSTAIRE helix. The N-terminal helix of cyclinA contacts the carboxy terminal lobe of CDK2.
These interactions will be detailed below.
III. CyclinA Induces Conformation Changes in CDK2
The interaction of CDK2 with cyclinA elicits an "open" position of the T-loop that allows target protein access to the catalytic active site cavity, bounded by the catalytic triad of residues Lys 33, Glu 51, and Asp 145, and ATP. This access affords phosphorylation of target residues by the kinase. Lys 33 is on an N-terminal β-strand/loop, Glu 51 is on the PSTAIRE α-helix, and Asp 145 is near the N-terminus of the T-loop.
A simulation of the conformational shifts of CDK2 structure that are elicited by the binding of cyclinA is presented here**. As the unbound (- cyclin) CDK2 conformation changes in response to cyclinA binding, please observe the profound shifts of a strand of the N-terminal β-sheet, the PSTAIRE α-helix, and the T-loop.
** Note: the presence of cyclinA is indicated in the simulation, but its 3-D coordinates are only estimated. Also, the movements are produced by linear interpolation, and the intermediate structures may not reflect actual folding states. This morph was created by first aligning PDB 1HCL with chain A of PDB 1FIN using the Superpose server (http://superpose.wishartlab.com/), saving the two chains as separate PDB files, and then employing the linear interpolation morpher provided by Karsten Theis: http://www.bioinformatics.org/pdbtools/morph2. In this process, gaps in the sequence of the kinase were generated. Please ignore these.
Repeating the above simulation as many times as desired, please focus on the following responses of CDK2 to cyclin binding:
- The movement of the catalytic residues (Lys 33, Glu 51, and Asp 145) that form the active site;
- The movement of the T-loop that opens access to the active site cavity, and the resulting accessible position of threonine160, the phosphorylation target of CDK2-activating kinase (CAK) that augments the activation of CDK2 by cyclinA;
- The melting of the α-helix at the amino end of the T-loop, permitting its large shift.
- The PSTAIRE helix rotates about 90° into the catalytic cleft, allowing for tight packing and catalytic interaction of Glu 51, which likely forms a salt bridge with Lys 33;
- The N-terminal lobe orientation changes relative to the PSTAIRE helix - the complexed N-terminal β-sheet sheet pivots ~14° from its position in uncomplexed CDK2 to open up the entrance to the catalytic cleft, allowing the PSTAIRE helix to pivot in.
Examination of a few cyclinA-CDK2 interactions exemplifies the weak bonds that drive the thermodynamically favored conformational shifts in CDK2. Focusing on the PSTAIRE region, which plays a central role at the cyclinA interface (Jeffrey, et al., 1995):
- Isoleucine 49 of the CDK2 PSTAIRE helix nestles into a hydrophobic pocket formed by the sidechains of Leu263 Phe 267, Leu 299, Leu 306, and the hydrocarbon portion of the Lys 266 sidechain of cyclinA.
- Another isoleucine of the PSTAIRE helix, Ile 52, engages in a van der Waals interaction with Phe 304 of cyclinA, and hydrophobic packing of Leu 54 and Phe 267 and Ala 307 of cyclinA is observed.
-
In complexed cyclinA-CDK2, a hydrogen bond network forms in the region adjacent to the PSTAIRE helix:
- the backbone carbonyls of Glu42 and Val44 are H-bond acceptors bonded to the H-bond donor N on the Lys 266 side chain of cyclinA;
- the backbone amide of Val 44 donates a hydrogen bond to the Glu 295 carboxylate of cyclinA;
- an additional hydrogen bond links the Lys 266 and Glu 295 side chains;
- The H-bonded network just illustrated is achieved by the conformation of the loop that extends from the PSTAIRE helix. This conformation is permitted by Gly 43, which adopts a backbone conformation (phi and psi angles) prohibited for other amino acids. Gly 43 of CDK2 and Lys 266 and Glu 295 of cyclinA are key elements of this network, and are highly conserved residues among CDKs and cyclins.
- Lys 56, near the C-terminus of the PSTAIRE helix, donates two hydrogen bonds to the Asp 305 side chain and the Thr 303 backbone carbonyl of cyclinA.
- Glu 57, near the N-terminus of the PSTAIRE helix, accepts a hydrogen bond from the Tyr 185 hydroxyl group of cyclinA.
These interactions illustrate the power of weak bonds in eliciting conformational changes wrought by protein-protein interactions. For more details about sterochemical interactions at the CDK2-cyclinA interface, the reader is referred to Jeffrey, et al. (1995).
IV. References
Abbas,Tarek,and Anindya Dutta (2006). CDK2-Activating Kinase (CAK). Cell Cycle 5:10, 1123-1124.
Bendris N, Lemmers B, Blanchard JM, Arsic N (2011). Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements. PLOS ONE. 6 (7): e22879. doi:10.1371/journal.pone.0022879.
Coverley D, Laman H, Laskey RA (2002). "Distinct roles for cyclins E and A during DNA replication complex assembly and activation". Nat. Cell Biol. 4 (7): 523–8. doi:10.1038/ncb813
Jeffrey, P.D., Russo, A.A., Polyak, K., Gibbs, E., Hurwitz, J., Massague, J., Pavletich, N.P. (1995). Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature 376: 313-320.
Pines J. (1994). The cell cycle kinases. Seminars in Cancer Biology. 5: 305-313.
Malumbres, M. (2014). Cyclin-dependent kinases. Genome Biol 15:122.
Schulze-Gahmen, U., De Bondt, H.L., Kim, S.H. (1996). High-resolution crystal structures of human cyclin-dependent kinase 2 with and without ATP: bound waters and natural ligand as guides for inhibitor design.J Med Chem 39: 4540-4546.
Woo RA, Poon RY (2003). "Cyclin-dependent kinases and S phase control in mammalian cells". Cell Cycle. 2 (4): 316–24. doi:10.4161/cc.2.4.468
Yam CH, Fung TK, Poon RY (2002). "Cyclin A in cell cycle control and cancer". Cell. Mol. Life Sci. 59 (8): 1317–26. doi:10.1007/s00018-002-8510-y