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The Role of the phospho CDK2/Cyclin A recruitment site in substrate recognition

DOI: 10.1074/jbc.M600480200 DOI Help

Authors: Kin-Yip Cheng (University of Oxford) , Martin E. M. Noble (University of Oxford) , Vicky Skamnaki (University of Oxford) , Nick R. Brown (University of Oxford) , Ed D. Lowe (University of Oxford) , Luke Kontogiannis (University of Oxford) , Kui Shen (Johns Hopkins University School of Medicine) , Philip A. Cole (Johns Hopkins University School of Medicine) , Giuliano Siligardi (Diamond Light Source) , Louise Johnson (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 281 , PAGES 23167-23179

State: Published (Approved)
Published: May 2006

Abstract: Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 Å from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the ?-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 ?m) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Medicine, Chemistry

Facility: ID14.1, ID23.1, ID29 at ESRF

Added On: 24/03/2010 00:31

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