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Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A
Edward D Lowe1, Ivo Tews, Kin Yip Cheng
1Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, Oxford OX1 3QU, UK.
Abstract:
Progression through S phase of the eukaryotic cell cycle is regulated by the action of the cyclin dependent protein kinase 2 (CDK2) in association with cyclin A. CDK2/cyclin A phosphorylates numerous substrates. Substrate specificity often employs a dual recognition strategy in which the sequence flanking the phospho-acceptor site (Ser.Pro.X.Arg/Lys) is recognized by CDK2, while the cyclin A component of the complex contains a hydrophobic site that binds Arg/Lys.X.Leu ("RXL" or "KXL") substrate recruitment motifs. To determine additional sequence specificity motifs around the RXL sequence, we have performed X-ray crystallographic studies at 2.3 A resolution and isothermal calorimetry measurements on complexes of phospho-CDK2/cyclin A with a recruitment peptide derived from E2F1 and with shorter 11-mer peptides from p53, pRb, p27, E2F1, and p107. The results show that the cyclin recruitment site accommodates a second hydrophobic residue either immediately C-terminal or next adjacent to the leucine of the "RXL" motif and that this site makes important contributions to the recruitment peptide recognition. The arginine of the RXL motif contacts a glutamate, Glu220, on the cyclin. In those substrates that contain a KXL motif, no ionic interactions are observed with the lysine. The sequences N-terminal to the "RXL" motif of the individual peptides show no conservation, but nevertheless make common contacts to the cyclin through main chain interactions. Thus, the recruitment site is able to recognize diverse but conformationally constrained target sequences. The observations have implications for the further identification of physiological substrates of CDK2/cyclin A and the design of specific inhibitors.
Insights
Cyclin-dependent kinase 2 (CDK2)/cyclin A recognizes diverse substrates through specific sequence motifs. This study reveals additional binding details, aiding in identifying new targets and designing inhibitors.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Progression through the S phase of the eukaryotic cell cycle is regulated by cyclin-dependent protein kinase 2 (CDK2) complexed with cyclin A.
- CDK2/cyclin A phosphorylates numerous substrates, employing a dual recognition strategy for specificity.
Purpose of the Study:
- To determine additional sequence specificity motifs around the RXL sequence for CDK2/cyclin A substrate recognition.
- To investigate the structural basis of substrate recruitment by CDK2/cyclin A.
Main Methods:
- X-ray crystallography at 2.3 Å resolution.
- Isothermal calorimetry measurements.
- Complex analysis of phospho-CDK2/cyclin A with recruitment peptides from E2F1, p53, pRb, p27, and p107.
Main Results:
- The cyclin recruitment site accommodates a second hydrophobic residue adjacent to the leucine in the RXL motif, contributing to peptide recognition.
- Arginine in the RXL motif contacts Glu220 on cyclin A; lysine in KXL motifs does not form ionic interactions.
- N-terminal sequences to RXL show no conservation but common main chain contacts to cyclin, indicating recognition of conformationally constrained sequences.
Conclusions:
- The CDK2/cyclin A recruitment site recognizes diverse target sequences through specific hydrophobic and main chain interactions.
- These findings are crucial for identifying new physiological substrates of CDK2/cyclin A and for designing specific inhibitors.