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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 A 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 gamma-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 microm) 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.
Highlights
Protein kinases catalyze the phosphorylation of serine, threonine, or tyrosine residues in target proteins
Many other CDK2 substrates that are important for cell cycle progression contain the recruitment motif RXL or KXL at a site that is remote from the site of phosphorylation (e.g. pRb, 2 The abbreviations used are: CDK, cyclin-dependent kinase; ATP␥S, adenosine 5Ј-O-3-thiotriphosphate; GST, glutathione S-transferase; AMPPNP, adenosine 5Ј-(,␥-imino)triphosphate; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol; PK, pyruvate kinase; PEP, phosphoenolpyruvate; LDH, lactate dehydrogenase; RMSD, root mean square deviation
Noting that with the insulin receptor tyrosine kinase domain (IRK), the bissubstrate was an effective inhibitor but that, in the crystal structure, the peptide component was located with more order than in the complex of the non-covalently linked peptide with AMPPNP, we have explored the use bis-substrate compounds in which the peptides are based on the model heptapeptide and the modified CDC6 peptide and in which the serine analogue amino-alanine is linked to ATP␥S via an acetyl bridge (Fig. 1)
Summary
Peptides—The heptapeptide substrate, the modified CDC6 peptide, and the recruitment HTL peptide HTLKGRRLVFDN, were synthesized by Dr G. The mass of the CDC6 peptide was confirmed by mass spectrometry and concentration determined by amino acid analysis. The cleaved peptide was further purified by reverse phase chromatography on a HPLC column (Jupiter 10u C5 by Phenomenex) followed by lyophilization. The lyophilized peptide was dissolved in aqueous solution, and its molecular mass confirmed by MALDI mass spectrometry. The amino-alanine-containing peptides were assembled on Wang resin using automated solid-phase peptide synthesis via the Fmoc strategy. Trifluoroacetic acid cleavage (with 5% phenol, 5% thioanisole, 5% water, 2.5% ethanedithiol, and 1% triisopropylsilane as scavengers), reverse phase HPLC, and lyophilization gave the bromoacetylated peptides confirmed by MALDI-TOF MS (the long peptide I, calculated [M] 3588, found [MϩH]ϩ 3588 Ϯ 1; the short peptide II, calculated [M] 993, found [MϩH]ϩ 993 Ϯ 1). 2.7-Å resolution were collected at ESRF station 14.1 (Table 1)
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