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J. Biol. Chem., Vol. 281, Issue 32, 23167-23179, August 11, 2006

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The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition^*

Kin-Yip Cheng, Martin E. M. Noble, Vicky Skamnaki, Nick R. Brown, Ed D. Lowe, Luke Kontogiannis, Kui Shen, Philip A. Cole, Giuliano Siligardi^¶, and Louise N. Johnson¹

From the Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, the Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the ^¶Diamond Light Source, Chilton, Oxon OX11 0QX, United Kingdom

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 K_m 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 (K_i = 0.83 nM) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (K_D 0.4 µM) 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.

Received for publication, January 17, 2006 , and in revised form, March 13, 2006.

The atomic coordinates and structure factors (code 2CCI (CDC6 peptide complex) and 2CCH (CDC6 bispeptide complex)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Medical Research Council, the European Community's Human Potential Programme under Contract HPRN-CT-2002-00252 (CAMKIN), the Wellcome Trust, and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratory of Molecular Biophysics, Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: 44-1865-275365; Fax: 44-1865-285353; E-mail: louise.johnson{at}biop.ox.ac.uk.

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