Title: The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition
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 γ-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 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. 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 γ-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 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. Protein kinases catalyze the phosphorylation of serine, threonine, or tyrosine residues in target proteins. They provide the signaling pathways by which extracellular signals (hormone, growth factor, etc.) are converted to intracellular responses through changes in metabolism or gene expression. There are over 500 protein kinases in the human genome (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanan S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6075) Google Scholar). Cross-talk between differently activated protein kinase signaling pathways is regulated by a variety of mechanisms that target the activity of an individual protein kinase to a select group of substrates. Most protein kinases exhibit specificity for a defined epitope around the site of phosphorylation, as first elaborated for cAMP-dependent kinase (2Kemp B.E. Bylund D.B. Huang T.S. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3448-3452Crossref PubMed Scopus (155) Google Scholar). Both the mitogen-activated protein kinase (MAPK) and the cyclin-dependent kinase (CDK) 2The abbreviations used are: CDK, cyclin-dependent kinase; ATPγS, adenosine 5′-O-3-thiotriphosphate; GST, glutathione S-transferase; AMPPNP, adenosine 5′-(β,γ-imino)triphosphate; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol; PK, pyruvate kinase; PEP, phosphoenolpyruvate; LDH, lactate dehydrogenase; RMSD, root mean square deviation.2The abbreviations used are: CDK, cyclin-dependent kinase; ATPγS, adenosine 5′-O-3-thiotriphosphate; GST, glutathione S-transferase; AMPPNP, adenosine 5′-(β,γ-imino)triphosphate; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol; PK, pyruvate kinase; PEP, phosphoenolpyruvate; LDH, lactate dehydrogenase; RMSD, root mean square deviation. families of protein kinases phosphorylate a serine or threonine residue as part of an (S/T)P motif that in CDK2 substrates is enhanced by a preference for basic residue in the P + 3 position (i.e. (S/T)PX(K/R)). The proline residues act as a powerful discriminator for other kinases (3Zhu G. Fujii K. Belkina N. Liu Y. James M. Herrero J. Shaw S. J. Biol. Chem. 2005; 280: 10743-10748Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) but may be insufficient to prevent cross-talk between CDK and MAPK pathways. Further mechanisms are utilized to enhance kinase specificity. CDK2 in complex with cyclin A is active during S phase of the cell cycle. Its activity is controlled in a temporal fashion through regulated transcription (and subsequent degradation) of the activatory cyclin subunits and the Kip/Cip p21 family of inhibitory proteins (4Harper J.W. Adams P.D. Chem. Rev. 2001; 101: 2511-2526Crossref PubMed Scopus (197) Google Scholar). Activation of CDK2/cyclin A requires phosphorylation on a threonine residue (Thr160 in the human CDK2 sequence) in the activation segment of the kinase by the cyclin-dependent kinase activating kinase CAK1. Levels of cyclin A remain high throughout S and G2 until cells enter mitosis where activation of the anaphase-promoting complex results in ubiquitination and destruction of cyclin A. At G2/M, levels of cyclin B rise, and CDK1/cyclin B initiates mitosis. The activities of the CDKs are governed by the cyclins relative to the phases of the cell cycle. The cyclins not only serve as activatory subunits but may also function in substrate recognition through direct interaction with substrates. Recruitment sites for phospho-CDK2/cyclin A (pCDK2/cyclin A) substrates that contain an RXL motif (where X is any amino acid) were first observed with the substrates p107 and p130 (5Ewen M.E. Faha B. Harlow E. Livingston D.M. Science. 1992; 255: 85-87Crossref PubMed Scopus (164) Google Scholar, 6Faha B. Ewen M. Tsai L.-H. Livingston D. Harlow E. Science. 1992; 255: 87-90Crossref PubMed Scopus (162) Google Scholar, 7Lees E. Faha B. Dulic V. Reed S.I. Harlow E. Genes Dev. 1992; 6: 1874-1885Crossref PubMed Scopus (360) Google Scholar, 8Hannon G.J. Demetrick D. Beach D. Genes Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar) and the CDK2 inhibitors p21 and p27 (9Zhu L. Harlow E. Dynlacht B.D. Genes and Dev. 1995; 9: 1740-1752Crossref PubMed Scopus (234) Google Scholar, 10Chen J. Saha P. Kornbluth S. Dynlacht B.D. Dutta A. Mol. Cell. Biol. 1996; 16: 4673-4682Crossref PubMed Scopus (273) Google Scholar). Many other CDK2 substrates that are important for cell cycle progression contain the recruitment motif RXLorKXLat a site that is remote from the site of phosphorylation (e.g. pRb, p53, E2F1, human papilloma virus (HPV) replication factor E1, CDC6, endomexin, Myt1, CDC25A (11Wohlschlegel J.A. Dwyer B.T. Takeda D.Y. Dutta A. Mol. Cell Biol. 2001; 21: 4868-4874Crossref PubMed Scopus (66) Google Scholar), the APC substrate-activating subunit Cdh1 (12Sorensen C.S. Lukas C. Kramer E.R. Peters J.M. Bartek J. Lukas J. Mol. Cell Biol. 2001; 21: 3692-3703Crossref PubMed Scopus (110) Google Scholar), p220NPAT (13Zhao J. Dynlacht B. Imai T. Hori T.-A. Harlow E. Genes Dev. 1998; 12: 456-461Crossref PubMed Scopus (181) Google Scholar), and BRAC2 (14Esashi F. Christ N. Gannon J. Liu Y. Hunt T. Jasin M. West S.C. Nature. 2005; 434: 598-604Crossref PubMed Scopus (352) Google Scholar)). Structural studies of pCDK2/cyclin A in complex with the p27Kip1 inhibitor (15Russo A.A. Jeffrey P.D. Patten A.K. Massague J. Pavletich N.P. Nature. 1996; 382: 325-331Crossref PubMed Scopus (788) Google Scholar) and with peptides from p107, pRb, E2F, p53 (16Lowe E.D. Tews I. Cheng K.Y. Brown N.R. Gul S. Noble M.E. Gamblin S.J. Johnson L.N. Biochemistry. 2002; 41: 15625-15634Crossref PubMed Scopus (144) Google Scholar) showed that the RXL recognition site is located on the cyclin A molecule at an exposed non-polar site containing the characteristic MRAIL cyclin sequence that is conserved in cyclins A and E (17Honda R. Lowe E.D. Dubinina E. Skamnaki V.T. Cook A. Brown N.R. Johnson L.N. EMBO J. 2005; 24: 452-463Crossref PubMed Scopus (104) Google Scholar). The recruitment site is some 40 Å from the catalytic site (as measured from the Cα of the serine at the substrate phosphorylation site and the Cα of the R of the RXL motif) (18Brown N.R. Noble M.E.M. Endicott J.A. Johnson L.N. Nature Cell Biology. 1999; 1: 438-443Crossref PubMed Scopus (470) Google Scholar). Mutation of the hydrophobic patch on cyclin A eliminates phosphorylation of substrates that require an RXL motif but not those CDK2 substrates, such as histone H1, that do not contain an RXL motif (19Schulman B. Lindstrom D.L. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10453-10458Crossref PubMed Scopus (301) Google Scholar). Fusion of an RXL motif to deletion mutants can restore activity (20Adams P.D. Li X. Sellars W.R. Baker K.B. Leng X. Harper J.W. Taya Y. Kaelin W.G. Mol. Cell Biol. 1999; 19: 1068-1080Crossref PubMed Scopus (161) Google Scholar). Recognition of the RXL motif by pCDK2/cyclin A or pCDK2/cyclin E may be sufficiently tight so that stable complexes can be observed as with p107, E2F, and the inhibitor p27Kip1. In other instances there is no stable association of the substrate with CDK2 but the integrity of the RXL (or KXL) motif has been shown to be essential for phosphorylation of target residues such as in the substrates pRb and p53 (19Schulman B. Lindstrom D.L. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10453-10458Crossref PubMed Scopus (301) Google Scholar, 21Driscoll B. T'ang A. Hu Y.-H. Yan C.L. Fu Y. Luo Y. Wu K.J. Wen S. Shi X.-H. Barsky L. Weinberg K. Murphree A.L. Fung Y.K. J. Biol. Chem. 1999; 274: 9463-9471Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Luciani M.G. Hutchins J.R.A. Zheleva D. Hupp T.R. J. Mol. Biol. 2000; 300: 503-518Crossref PubMed Scopus (61) Google Scholar). Paradoxically stronger physical association between the CDK/cyclin and the substrate does not necessarily lead to a more efficient phosphorylation than weaker interaction, as shown with studies E2F/DP1 phosphorylation (23Guida P. Zhu L. Biochim. Biophys. Res. Commun. 1999; 258: 596-604Crossref PubMed Scopus (9) Google Scholar). Mutagenesis experiments with p107 and kinetic experiments with model peptides have shown that one of the roles of the RXL motif is to make a poor substrate with suboptimal consensus phosphorylation motif an effective pCDK2/cyclin A substrate (24Leng X. Noble M.E.M. Adams P.D. Qin J. Harper J.W. Mol. Cell Biol. 2002; 22: 2242-2254Crossref PubMed Scopus (70) Google Scholar, 25Stevenson-Lindert L.M. Fowler P. Lew J. J. Biol. Chem. 2003; 278: 50956-50960Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). RXL-containing peptides are effective inhibitors of the activity of pCDK2/cyclin A against RXL-containing substrates and have potential therapeutic applications (26Andrews M.J. McInnes C. Kontopidis G. Innes L. Cowan A. Plater A. Fischer P.M. Org. Biomol. Chem. 2004; 2: 2735-2741Crossref PubMed Scopus (55) Google Scholar, 27Chen Y.-N.P. Sharma S.K. Ramsey T.M. Liang L. Martin M.S. Baker K. Adams P.D. Bair K.W. Kaelin W.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4325-4329Crossref PubMed Scopus (306) Google Scholar, 28Mendoza N. Fong S. Marsters J. Koeppen H. Schwall R. Wickramasinghe D. Cancer Res. 2003; 63: 1020-1024PubMed Google Scholar). The structural mechanism by which the RXL recognition site on cyclin A promotes substrate phosphorylation by pCDK2 is not understood. There are no conformational changes when the RXL site is occupied (16Lowe E.D. Tews I. Cheng K.Y. Brown N.R. Gul S. Noble M.E. Gamblin S.J. Johnson L.N. Biochemistry. 2002; 41: 15625-15634Crossref PubMed Scopus (144) Google Scholar, 18Brown N.R. Noble M.E.M. Endicott J.A. Johnson L.N. Nature Cell Biology. 1999; 1: 438-443Crossref PubMed Scopus (470) Google Scholar). The most likely mechanism is that the RXL motif serves as an entropic effector, localizing the CDK close to the substrate leading to an increase in the local substrate concentration (19Schulman B. Lindstrom D.L. Harlow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10453-10458Crossref PubMed Scopus (301) Google Scholar) but mechanisms that envisage a more direct participation between the RXL and catalytic site are also possible (29Takeda D.Y. Wohlschlegel J.A. Dutta A. J. Biol. Chem. 2001; 276: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To explore the relationship between the recruitment site and catalytic site, we have carried out structural and kinetic studies with a substrate peptide that contains the recruitment motif. We based our peptide on the naturally occurring CDC6 protein that contains a phosphorylation site twenty amino acids N-terminal to the RXL site. To make direct comparison with the optimal heptapeptide (HHASPRK) identified from library screening (30Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwica-Worms H. Cantley L.C. Curr. Biol. 1994; 4: 973-982Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar) and which we used in previous structural studies, we changed the CDC6 peptide sequence from the wild-type sequence: 71PPCSPPKQGKKENGPPHSHTLKGRRLVFDN100 to 71HHASPRKQGKKENGPPHSHTLKGRRLVFDN100, where the sequences around the phosphorylated serine and the RXL motif are highlighted in bold. In a mechanism-based approach to protein kinase inhibitors, bis-substrate compounds have been introduced in which the γ-phosphate of ATP is covalently attached by a linker to the amino analogue of the OH acceptor amino acid located in a peptide of cognate substrate sequence (31Ablooglu A.J. Till J.H. Kim K. Parang K. Cole P.A. Hubbard S.R. Kohanski R.A. J. Biol. Chem. 2000; 275: 30394-30398Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 32Parang K. Till J.H. Ablooglu A.J. Kohanski R.A. Hubbard S.R. Cole P.A. Nat. Structural Biology. 2001; 8: 37-41Crossref PubMed Scopus (198) Google Scholar, 33Hines A.C. Cole P.A. Bioorg. Med. Chem. Lett. 2004; 14: 2951-2954Crossref PubMed Scopus (32) Google Scholar). Noting that with the insulin receptor tyrosine kinase domain (IRK), the bis-substrate was not only 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 (hereafter referred to as bispeptides) 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 ATPγS via an acetyl bridge (Fig. 1). Structural, kinetic, and binding studies with the substrate peptides and the bispeptide inhibitors show that the presence of the recruitment motif significantly increases affinity for substrates and inhibitors, respectively. Kinetic studies show that there is an obligation for a covalent path between the catalytic site and the RXL motif, but no direct stereochemical path is observed in the crystal studies. A model is proposed based on localization effects and a flexible linker, which must be greater than a specified minimal length (about 15-16 residues), to explain the nanomolar potency of the CDC6 bispeptide inhibitor. Peptides—The heptapeptide substrate, the modified CDC6 peptide, and the recruitment HTL peptide HTLKGRRLVFDN, were synthesized by Dr. G. Bloomberg (University of Bristol Peptide Synthesis Laboratory) and purified by HPLC. The mass of the CDC6 peptide was confirmed by mass spectrometry and concentration determined by amino acid analysis. The p107 peptide (Fig. 1) was expressed as a GST fusion protein with the vector pGEX-6P from B834 DE3 pLyS cells at 18 °C overnight, purified on a glutathione-Sepharose fast flow 4B affinity chromatography column (Amersham Biosciences), and the GST tag cleaved by 3C protease. 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. Its concentration was determined by amino acid analysis. Synthesis of ATP-conjugated Peptides I and II—Peptides were synthesized following the same strategy as described previously (33Hines A.C. Cole P.A. Bioorg. Med. Chem. Lett. 2004; 14: 2951-2954Crossref PubMed Scopus (32) Google Scholar). The amino-alanine-containing peptides were assembled on Wang resin using automated solid-phase peptide synthesis via the Fmoc strategy. The allyloxycarbonyl (alloc) protection of the side chain of amino-alanine was removed by a 2-h treatment under nitrogen with 5 eq of Pd(PPh3)4, 10 eq of N-methylmorpholine, and 20 eq of acetic acid in chloroform. The side chain of amino-alanine was then bromoacetylated in dimethylformamide for 30 min using symmetric anhydride formed by a 10-min mixing of 10 eq of di-isopropylcarbodiimide and 5 eq of bromoacetic acid in methylene chloride. 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 [M+H]+ 3588 ± 1; the short peptide II, calculated [M] 993, found [M+H]+ 993 ± 1). Treatment of the bromoacetylated peptides with ATPγS (1.5 eq; Roche Applied Science) in 0.1 m ammonium acetate buffer (pH 7.0) (∼10 mg peptide in 1 ml of solution) overnight at room temperature followed by gel filtration using Bio-Gel P-2 Gel (∼15 mg purified over ∼50 ml resin) in 0.1 m ammonium acetate buffer (pH 7.0) and lyophilization gave the desired ATP-conjugated peptides confirmed by ESI MS (the long peptide I, calculated [M] 4034, found [M+5H]5+ 808, [M+4H]4+ 1009, [M+3H]3+ 1346, [M+2H]2+ 2018, [M+H]+ 4034 ± 1 (reconstructed); the short peptide II, calculated [M] 1431, found [M+Li]+ 1437 ± 1). Crystallization and Crystal Structure of pCDK2/Cyclin A-CDC6 Peptide Complex—Phospho-CDK2/cyclin A was prepared as previously described (18Brown N.R. Noble M.E.M. Endicott J.A. Johnson L.N. Nature Cell Biology. 1999; 1: 438-443Crossref PubMed Scopus (470) Google Scholar). CDC6 peptide (0.5 mm) was mixed with 10 mg/ml purified pCDK2/cyclin A (0.15 mm) together with 1 mm AMPPNP and left on ice for 30 min. Crystals were grown by the sitting drop vapor diffusion method in which 1 μl of the protein sample was mixed with 1 μl of precipitant from the reservoir containing 10-17% (v/v) PEG monomethylether 5000, 0.2 m ammonium sulfate, and 0.1 m MES pH 5.6 at 4 °C. Crystals were briefly cryoprotected with 20% (v/v) glycerol solution (in precipitant solution), frozen in liquid nitrogen, and diffraction data collected to 2.4-Å resolution at ESRF station 14.1. The data were processed using MOSSFLM and CCP4 programs (34Leslie A.G.W. MOSFLM. Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography. Daresbury Laboratory, Warrington1992Google Scholar, 35P4 CC Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar) and a difference electron density map calculated. There was clear binding of the CDC6 peptide at the RXL site for residues HTLKGRRLVFDN and for AMPPNP at the catalytic site, but there was no clear density for a peptide at the catalytic site. Previously we had observed peptide binding at the CDK2 catalytic site by transferring the crystals, which had been grown with lithium sulfate as the precipitant, to a non-polar solvent in order to strengthen polar interactions (18Brown N.R. Noble M.E.M. Endicott J.A. Johnson L.N. Nature Cell Biology. 1999; 1: 438-443Crossref PubMed Scopus (470) Google Scholar). We reasoned that the ammonium sulfate in the present crystallization condition, albeit at a concentration of only 0.2 m, could have affected binding of the CDC6 peptide to the catalytic site. Crystals of the ternary complex were grown as described above and back-soaked into a solution that was the same as the original conditions but without ammonium sulfate and which contained 10 mm CDC6 peptide, 3 mm AMPPNP, and 5 mm MgCl2 (to facilitate nucleotide binding) for 30 min at 4 °C before brief cryoprotection with 20% (v/v) glycerol solution and freezing in liquid nitrogen. Data to 2.7-Å resolution were collected at ESRF station 14.1 (Table 1).TABLE 1Data collection and refinement statistics for pCDK2/cyclin A-CDC6 peptide and pCDK2/cyclin A-CDC6 bispeptide complexesCDC6 peptide: cocrystallized (0.5 mm)CDC6 peptide: back-soak (10 mm)CDC6 bispeptide: back-soak (0.58 mm with 5 mm Mg2+)CDC6 bispeptide: back-soak (0.58 mm with no Mg2+)Data collectionBeamlineESRF station ID 14.1ESRF station ID 14.1ESRF station ID 29ESRF station ID 23.1Space groupP212121P212121P212121P212121Unit cell (Å)a = 74.1a = 74.4a = 74.8a = 74.53b = 113.6b = 114.4b = 114.7b = 114.48c = 181.3c = 170.7c = 181.5c = 181.30Resolution (highest range) (Å)2.4 (2.53-2.4)2.7 (2.85-2.7)2.4 (2.53-2.4)1.7 (1.79-1.7)Observations142,61886,854272,633612,349Unique reflections58,62639,62961,844167,702% Completeness (highest resolution)97.3 (96.7)97.6 (98.5)99.9 (99.9)98.0 (91.5)RsymaRsym = ΣhΣi<Ih>—Ihi/ΣIhi where Ihi is the intensity of the ith observation of unique reflection h. (highest resolution)0.075 (0.131)0.116 (0.313)0.101 (0.358)0.058 (0.368)Mean I/σI (highest resolution)12 (5.3)10.4 (2.2)11.7 (4.8)15.1 (2.8)Refinement statisticsProtein and ligand atoms9206925791979481No. of waters1961347361045Resolution range (Å)23.4-2.440.3-2.797.1-2.496.97-1.7RconvbRconv = Σ∥Foh|—|Fch∥/Σ|Foh| where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h.0.200.260.1750.150RfreecRfree is equivalent to Rconv but is calculated using a 5% disjoint set of reflections excluded from the least squares refinement.0.250.320.2320.183RMSD bond length (Å)0.0180.0080.0220.017RMSD bond angle (°)1.551.151.91.59Mean protein temperature factors (Å2)Overall36.237.738.624.4Chain A CDK227.836.624.817.1Chain B cyclin A25.136.122.015.7Chain C CDK246.938.456.831.2Chain D cyclin A42.437.849.326.5Chain E substrate or recruitment peptide49.354.842.330.6Chain F recruitment peptide63.644.359.141.6Chain G AMPPNP or ATP43.173.470.2Chain H substrate peptide55.4Chain I recruitment peptide63.3Chain J AMPPNP41.1Chain W waters39.228.936.032.3a Rsym = ΣhΣi<Ih>—Ihi/ΣIhi where Ihi is the intensity of the ith observation of unique reflection h.b Rconv = Σ∥Foh|—|Fch∥/Σ|Foh| where Foh and Fch are the observed and calculated structure factor amplitudes for reflection h.c Rfree is equivalent to Rconv but is calculated using a 5% disjoint set of reflections excluded from the least squares refinement. Open table in a new tab The crystals have space group P212121 with 2 molecules of pCDK2/cyclin A per asymmetric unit. The c axis shrank nearly 10 Å during the back-soak procedure, and the crystal diffracted less well. The structure of the back-soaked crystal was solved by molecular replacement with MOLREP (35P4 CC Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar) followed by rigid body refinement using REFMAC5 (36Murshudov G.N. Vagen A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). The sigmaA-weighted 2Fo - Fc electron density map showed the presence of the CDC6 peptide at both the catalytic site and recruitment site. The model was refined with cycles of restrained refinement and manual rebuilding using O (37Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). This refinement process was intercalated with 4 rounds of simulated annealing and energy optimization (CNS) (38Brünger A.T. X-PLOR: Version 3.8; A System for Protein Crystallography and NMR. Yale University Press, New Haven1996Google Scholar) using an initial temperature of 2500 K with subsequent cooling step of 25 K per cycle of dynamics. Crystallization and Crystal Structure of the pCDK2/Cyclin A-CDC6 Bispeptide Complex—The complex was crystallized using the sitting drop vapor diffusion method in which 1 μl of pCDK2/cyclin A (18 mg/ml; 0.28 mm) containing 0.29 mm CDC6 bispeptide was mixed 1 μl of reservoir solution containing 13% PEG monomethylether 5000, 0.2 m ammonium sulfate, 0.1 m citrate/acetate buffer pH 5.6 at 4 °C. Three weeks later a crystal was taken and back-soaked in a ammonium sulfate-free solution containing 13% PEG monomethylether 5000, 0.1 m citrate/acetate buffer pH 5.6, 15% glycerol, 5 mm MgCl2, 0.58 mm bispeptide overnight before freezing with liquid nitrogen and data collection (Table 1). In a second experiment, magnesium was omitted from the final soak so as to minimize any possible ATPase activity. The pCDK2/cyclin A (18 mg/ml) was co-crystallized with 0.29 mm bispeptide at 4 °C with 14% PEG monomethylether 5000, 0.2 m ammonium sulfate, 0.1 m citrate/acetate buffer pH 5.6. Ten weeks later, a crystal was taken and back-soaked in 14% PEG monomethylether 5000, 0.1 m citrate/acetate buffer, 15% glycerol, 0.58 m bispeptide for 4-5 min before freezing with liquid nitrogen and data collection (Table 1). Both experiments showed binding at the recruitment site for a dodecamer peptide and weak binding at the catalytic site in which only the modified ATP was clearly located. Two conformations were visible for the ribose and triphosphate in both CDK2 molecules and also two conformations for residues Asp145 and Leu148. The structure for the 1.7-Å data (Table 1) for the pCDK2/cyclin A-bispeptide complex, including the recruitment peptide, ATP, and waters assigned by ARP/wARP, was further refined using TLS (a procedure that allows correlation of internal torsional motion with overall molecular motion in crystals) and isotropic B factors, followed by TLS with anisotropic B factors with strong restraints toward isotropic values, and finally with TLS and anisotropic B values and hydrogen atoms as determined by REFMAC. At each stage the Rfree value was carefully monitored. The refinement statistics (Table 1) support the incorporation of anisotropic B values and hydrogen atoms. Kinetics—Pyruvate kinase (PK), lactate dehydrogenase (LDH), phosphoenolpyruvate (PEP), ATP, NADH, and other chemicals were obtained from Sigma. The pCDK2/cyclinA concentration was determined according to Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Phosphorylation of the peptides was measured by a spectrophotometric assay in which ADP production was coupled to the NADH oxidation by PK and LDH as previously described (40Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (132) Google Scholar, 41Cook F.N. Neville M.E. Vrana K.E. Hartl F.T. Roskowski R. Biochemistry. 1982; 21: 5794-5799Crossref PubMed Scopus (343) Google Scholar) with minor modifications. The molar extinction coefficient for NADH was assumed to be 6220 m−1 cm−1 at 340 nm. All reactions were performed at 30 °C. The assay mixture (volume 0.25 ml) for measurement of pCDK2/cyclin A activities with the heptapeptide contained 30 units/ml LDH, 12 units/ml PK, 1 mm PEP, 0.128 mm NADH, 0.5 mg/ml bovine serum albumin, 50 mm HEPES (pH 7.5), 150 mm NaCl, 10 mm KCl, 10 mm MgCl2, 2mm dithiothreitol with the heptapeptide substrate concentration between 0.106-2.13 mm. After incubation for 1-2 min, the reaction was initiated by the simultaneous addition of 0.74 mm ATP and pCDK2/cyclin A (0.48 μg/ml). Aliquots (0.08 ml) were withdrawn at 3, 6, and 9 min and transferred into 0.08 ml of sodium dodecyl sulfate (0.2%). Assays with the heptapeptide substrate were also performed in the presence of 0.75 mm of the recruitment HTL peptide. Control reactions in the absence of peptide substrate were used to monitor any ATPase activity. Reaction rates were found to be linear with enzyme concentration. Measurements with the CDC6 peptide were made using similar conditions. CDC6 peptide concentrations were varied from 0.032-0.48 mm, and reactions were stopped at 13 min with 20 mm triethanolamine pH 8.0, 200 mm EDTA to avoid precipitation of the longer peptides that occurred with SDS. Kinetic data were analyzed with the nonlinear regression program GraFit (42Leatherbarrow R.J. GrafFit Version 3.0. Erithakus Software, Staines, UK1992Google Scholar). The assay mixture for inhibition measurements with the CDC6 bispeptide against the heptapeptide and the p107 peptide substrates was as above except the reaction volumes were 0.08 ml and NADH 0.14 mm. Concentrations of the CDC6 bispeptide inhibitor were 58 nm and 116 nm in
Publication Year: 2006
Publication Date: 2006-08-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 91
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