Structural prediction of the interaction of the tumor suppressor p27KIP1 with cyclin A/CDK2 identifies a novel catalytically relevant determinant

Abstract

BackgroundThe cyclin-dependent kinase 2 (CDK2) together with its cyclin E and A partners is a central regulator of cell growth and division. Deregulation of CDK2 activity is associated with diseases such as cancer. The analysis of substrates identified S/T-P-X-R/K/H as the CDK2 consensus sequence. The crystal structure of cyclin A/CDK2 with a short model peptide supports this sequence and identifies key interactions. However, CDKs use additional determinants to recognize substrates, including the RXL motif that is read by the cyclin subunits. We were interested to determine whether additional amino acids beyond the minimal consensus sequence of the well-studied substrate and tumor suppressor p27KIP1 were relevant for catalysis.ResultsTo address whether additional amino acids, close to the minimal consensus sequence, play a role in binding, we investigate the interaction of cyclin A/CDK2 with an in vivo cellular partner and CDK inhibitor p27KIP1. This protein is an intrinsically unfolded protein and, in particular, the C-terminal half of the protein has not been accessible to structural analysis. This part harbors the CDK2 phosphorylation site. We used bioinformatics tools, including MODELLER, iTASSER and HADDOCK, along with partial structural information to build a model of the C-terminal region of p27KIP1 with cyclin A/CDK2. This revealed novel interactions beyond the consensus sequence with a proline and a basic amino acid at the P + 1 and the P + 3 sites, respectively. We suggest that the lysine at P + 2 might regulate the reversible association of the second counter ion in the active site of CDK2. The arginine at P + 7 interacts with both cyclin A and CDK2 and is important for the catalytic turnover rate.ConclusionOur modeling identifies additional amino acids in p27KIP1 beyond the consensus sequence that contribute to the efficiency of substrate phosphorylation.

Targeted Induction of Endogenous VDUP1 by Small Activating RNA Inhibits the Growth of Lung Cancer Cells

“…p27 and p53 are well-known cell-cycle regulators. p27 binds and inhibits cyclin A- or cyclin E-associated cyclin-dependent kinase 2 activity [ 19 ]. p53 is known as an important regulator of G2/M transition, and it induces cell-cycle arrest by inhibition of cyclin-dependent kinase 1 and repression of the cyclin B1 gene expression [ 20 ].…”

Machine Learning-Based Scoring Functions, Development and Applications with SAnDReS

“…(3), we have the residues participating in most of the intermolecular contacts for the crystallographic structures in the CDK dataset. We see the preponderance of the electrostatic intermolecular interactions with the participation of charged residues Glu 12, Lys 33, Glu 81, His 84, Gln 85, Asp 86, Asn 132 and Asp 145 [91][92][93][94][95][96].…”

p27Kip1, an Intrinsically Unstructured Protein with Scaffold Properties

“…Referring to the relations of p27 with Cyclin/CDK complexes, in addition to the mechanism of interaction of p27 with Cyclin A/CDK2 elucidated in detail (here described in paragraph 2.1 and summarized in Figure 2 B), Li and colleagues [ 47 ] highlighted, mostly by bioinformatic tools, further regions, far-off the KID, that could be involved in Cyclin A/CDK2 stabilization into an active state and in proper substrate recognition, in both of which the R194-p27 (located at +7 from CDK2 phosphorylation site, i.e., T187) may play a crucial role. In detail, R194 seems to establish H-bonds with P272 of Cyclin A and E42 of CDK2, located at the binding interface between cyclin A and CDK2.…”

“…It is important to stress, however, that the importance of R194 has been experimentally validated by demonstrating that a peptide (180–194 of p27 sequence) including R194 is a better substrate of CyclinA/CDK2 than a similar peptide with alanine in 194 position [ 47 ]. Thus, further studies are clearly necessary for confirming the precise role of R194 in the ternary Cyclin/CDK/p27 complexes.…”