Solution NMR Study of the Monomeric Form of p13 Protein Sheds Light on the Hinge Region Determining the Affinity for a Phosphorylated Substrate
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m111741200
ISSN1083-351X
AutoresBenoı̂t Odaert, Isabelle Landrieu, Klaas Dijkstra, Gea K. Schuurman‐Wolters, Peter Casteels, Jean‐Michel Wieruszeski, Dirk Inzé, Ruud M. Scheek, Guy Lippens,
Tópico(s)Signaling Pathways in Disease
ResumoCyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal β-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13suc1 CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the β-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13suc1 and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal β-sheet involved in domain swapping and protein interactions. Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal β-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13suc1 CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the β-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13suc1 and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal β-sheet involved in domain swapping and protein interactions. Progression through the cell cycle is tightly regulated at all levels with a central role for the CDK 1The abbreviations used are: CDKcyclin-dependent kinaseCdccell division control proteinCKScyclin-dependent kinase subunitCSIchemical shift indexHSQCheteronuclear single quantum spectroscopyNOEnuclear Overhauser effectNOESYNOE spectroscopyTOCSYtotal correlation spectroscopyPin1protein interacting with NIMASCFSkp1·Cdc53·Fbox complexsucsuppressor of cell cycle block complex. Activation of the kinase requires binding of a cyclin protein (1.Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar) as well as phosphorylations and dephosphorylations of specific residues (2.Lew D.J. Kornbluth S. Curr. Opin. Cell Biol. 1996; 8: 795-804Crossref PubMed Scopus (280) Google Scholar). Additionally a small subunit called cyclin-dependent kinase subunit (CKS) is essential for cell cycle progression (3.Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar), and the cell is unable to enter or exit mitosis depending on the time at which the CKS is removed (4.Patra D. Dunphy W.G. Genes Dev. 1996; 10: 1503-1515Crossref PubMed Scopus (81) Google Scholar). Although this small protein does not modulate directly the catalytic function of the CDK complex, it is involved in the interaction with regulators of the kinase complex (5.Patra D. Wang S.X. Kumagai A. Dunphy W.G. J. Biol. Chem. 1999; 274: 36839-36842Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). CKS enhances the Cdc25 phosphatase phosphorylation by the CDK complex (5.Patra D. Wang S.X. Kumagai A. Dunphy W.G. J. Biol. Chem. 1999; 274: 36839-36842Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), binds to the phosphorylated anaphase-promoting complex responsible for the cyclin degradation that is necessary to exit mitosis (6.Patra D. Dunphy W.G. Genes Dev. 1998; 12: 2549-2559Crossref PubMed Scopus (122) Google Scholar), and is required for SCFskp2-mediated ubiquitinylation of the phosphorylated p27kip2 (7.Ganoth D. Bornstein G. Ko T.K. Larsen B. Tyers M. Pagano M. Hershko A. Nat. Cell Biol. 2001; 3: 321-324Crossref PubMed Scopus (422) Google Scholar). We have recently shown that the Schizosaccharomyces pombe p13suc1protein binds via its anion-binding site to a Cdc25 phosphatase peptide in a phosphorylation-dependent manner (8.Landrieu I. Odaert B. Wieruszeski J.-M. Drobecq H. Inzé D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). cyclin-dependent kinase cell division control protein cyclin-dependent kinase subunit chemical shift index heteronuclear single quantum spectroscopy nuclear Overhauser effect NOE spectroscopy total correlation spectroscopy protein interacting with NIMA Skp1·Cdc53·Fbox complex suppressor of cell cycle block A potential binding site for the phosphorylated substrate was suggested by the presence of a sulfate anion in the crystal structure of CKSHs2 (9.Parge H.E. Arvai A.S. Murtari D. Reed S.I. Tainer J.A. Science. 1993; 262: 387-395Crossref PubMed Scopus (109) Google Scholar). Several other structures have been solved, including the human CKSHs1 (10.Arvai A.S. Bourne Y. Hickey M.J. Tainer J.A. J. Mol. Biol. 1995; 249: 835-842Crossref PubMed Scopus (68) Google Scholar), the S. pombe p13suc1(11.Endicott J.A. Noble M.E. Garman E.F. Brown N. Rasmussen B. Nurse P. Johnson L.N. EMBO J. 1995; 14: 1004-1014Crossref PubMed Scopus (67) Google Scholar, 12.Bourne Y. Arvai A.S. Bernstein S.L. Watson M.H. Reed S.I. Endicott J.E. Noble M.E. Johnson L.N. Tainer J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10232-10236Crossref PubMed Scopus (67) Google Scholar, 13.Khazanovich N. Bateman K.S. Chernaia M. Michalak M. James M.N.G. Structure. 1996; 4: 299-309Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and the Saccharomyces cerevisiae CKS1 (14.Bourne Y. Watson M.H. Arvai A.S. Bernstein S.L. Reed S.I. Tainer J.A. Struct. Fold. Des. 2000; 8: 841-850Abstract Full Text Full Text PDF Scopus (35) Google Scholar), and all contained the conserved cluster of basic residues that form the potential anion-binding site. A recent NMR study has confirmed that those residues map to the interaction site with the phospho-(Ser/Thr)-Pro motif on the ligand (8.Landrieu I. Odaert B. Wieruszeski J.-M. Drobecq H. Inzé D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The conserved CKS fold further contains a four-stranded β-sheet flanked by two α-helices where the four-stranded β-sheet has on one side a patch of conserved hydrophobic residues that bind tightly to the C-terminal domain of the kinase as revealed by the crystal structure of the human CKSHs1·CDK2 complex (15.Bourne Y. Watson M.H. Hickey M.J. Holmes W. Rocque W. Reed S.I. Tainer J.A. Cell. 1996; 84: 863-874Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Several crystal structures have revealed that the CKS monomers can form a dimer by exchange of their respective C-terminal β-strands (β4) (9.Parge H.E. Arvai A.S. Murtari D. Reed S.I. Tainer J.A. Science. 1993; 262: 387-395Crossref PubMed Scopus (109) Google Scholar, 12.Bourne Y. Arvai A.S. Bernstein S.L. Watson M.H. Reed S.I. Endicott J.E. Noble M.E. Johnson L.N. Tainer J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10232-10236Crossref PubMed Scopus (67) Google Scholar, 13.Khazanovich N. Bateman K.S. Chernaia M. Michalak M. James M.N.G. Structure. 1996; 4: 299-309Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 14.Bourne Y. Watson M.H. Arvai A.S. Bernstein S.L. Reed S.I. Tainer J.A. Struct. Fold. Des. 2000; 8: 841-850Abstract Full Text Full Text PDF Scopus (35) Google Scholar). The mechanism of this domain swapping remains unknown, and no consensus model has yet been achieved. Mutation studies (16.Schymkowitz J.W.H. Rousseau F. Irvine L.R. Itzhaki L.S. Structure. 2000; 8: 89-100Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 17.Schymkowitz J.W.H. Rousseau F. Itzhaki L.S. J. Mol. Biol. 2000; 301: 199-204Crossref PubMed Scopus (16) Google Scholar) and molecular dynamics simulations of protein unfolding (18.Alonso D.O.V. Alm E. Daggett V. Structure. 2000; 8: 101-110Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) of the S. pombe p13suc1 CKS monomeric protein suggest that dimerization occurs in the early events of the folding process via an intermediate state or even imply in vivo chaperone molecules not yet characterized. However, it is clear that this domain swapping requires only a minimal conformational change in the hinge region preceding the β4-strand. Moreover, the conservation of this H XPEPH motif among the different members of the CKS family argues for a biologically relevant role that might be related to a conformational control of the CKS association with the CDK complexes (19.Endicott J.A. Nurse P. Structure. 1995; 3: 321-323Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 20.Pines J. Curr. Biol. 1996; 6: 1399-1402Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Indeed, as this hinge region switches from a turn (in the closed globular form) to an extended conformation (in the open exchanged form), the protein in its dimeric form can no longer interact with the kinase (15.Bourne Y. Watson M.H. Hickey M.J. Holmes W. Rocque W. Reed S.I. Tainer J.A. Cell. 1996; 84: 863-874Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Very recently it was shown that single point mutations of residues as far as 20 Å from the hinge motif or, alternatively, ligand binding can equally shift the equilibrium between both forms toward the monomeric form (21.Schymkowitz J.W.H. Rousseau F. Wilkinson H.R. Friedler A. Itzhaki L.S. Nat. Struct. Biol. 2001; 8: 888-892Crossref PubMed Scopus (50) Google Scholar). Whereas these last results suggest that the strain accumulated throughout the protein is actively used to enhance ligand binding, many questions remain, one of the most important being the uncertainty on the solution conformation of this β4-strand. Small angle x-ray scattering studies on the native S. pombep13suc1 CKS monomeric protein in solution yielded an unexpected large gyration radius, suggesting that CKS even in its monomeric form is at least partially in an open conformation with this C-terminal β-strand protruding in solution (22.Birck C. Vachette P. Welch M. Swaren P. Samama J.P. Biochemistry. 1996; 35: 5577-5585Crossref PubMed Scopus (17) Google Scholar). In the same S. pombe p13suc1 CKS protein, mutation of the first proline residue of the hinge region to an alanine (p13P90A) has been reported to prevent the swapping process, to increase the protein stability, and to simplify the folding kinetics (23.Rousseau F. Schymkowitz J.W.H. Sánchez del Pino M. Itzhaki L.S. J. Mol. Biol. 1998; 284: 503-519Crossref PubMed Scopus (32) Google Scholar). To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13suc1 and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. We therefore continued our NMR analysis to characterize structurally the CKS protein in solution and to investigate the role of the proline residue Pro-90 (bold underlined) in the hinge region (motif H X PEPH). We found that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal β-sheet involved in domain swapping and protein interactions. The flexibility imposed by the loaded molecular spring that is the hinge region can provide for the diversity in binding partners that the CKS protein has to recognize during the cell cycle. The p13suc1 gene was cloned at the NdeI and EcoRI sites of the T7 promotor-based vector pRK172 (3.Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar). The proline mutation into alanine at residue position 90 was obtained by PCR using primers containing a single point mutation converting the proline GCA codon into an alanine CCA codon. For production of uniformly 15N,13C-labeled p13suc1 proteins, BL21 cells hosting the plasmid were grown in M9 minimal medium with 15NH4Cl (1 g liter−1) and [13C]glucose (2 g liter−1) (Cambridge Isotope Laboratories, Cambridge, MA) to A600 = 0.6–1.0 and induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside for 2 h. After centrifugation for 10 min at 5000 × g, the cells were resuspended in Tris-HCl buffer (50 mm, pH 8) containing 1 mm phenylmethylsulfonyl fluoride, 1 mm MgCl2, and traces of DNase and RNase and disrupted with a French press. After centrifugation of the solution for 2 h at 4 °C at 17,000 × g, the supernatant was dialyzed overnight at 4 °C against the Tris-HCl buffer. Proteins were purified with a Q anion chromatography-Sepharose column equilibrated in the Tris-HCl buffer with a NaCl elution gradient (0–0.5 m) followed by a gel filtration step with a G75 column equilibrated with 50 mm Tris-HCl, NaCl buffer. At each step, fractions containing the pure monomeric form of p13suc1 proteins as checked by native and SDS-polyacrylamide gels were pooled together and concentrated. The dimeric fraction represented less than 5% of the wild-type protein and was separated from the monomeric fraction during the purification process. NMR samples were prepared by dialysis of the protein solution against 100 mm NaCl, 50 mmNa2HPO4/NaH2PO4solution (pH 6.8) in 5%:95% D2O/H2O and concentrated by centrifugation to a final protein concentration of 2 mm (Vivaspin 5 kDa). NMR data were recorded at 20 °C on a Bruker DMX600 (Pasteur Lille) and on a Varian Inova 600 (Groningen) spectrometer equipped with a triple resonance 5-mm probe with a z gradient coil. Sequential backbone resonance assignments of p13suc1 and p13P90A protein were achieved using the following pairs of triple resonance experiments: HNCA/HN(CO)CA, HNCO/HN(CA)CO, CBCANH/CBCA(CO)NH, or HNCACB and combination of sensitivity enhancement versions of15N-edited HSQC-NOESY/HSQC-TOCSY (24.Grzesiek S. Bax A. Hu J.S. Kaufman J. Palmer I. Stahl S.J. Tjandra N. Wingfield P.T. Protein Sci. 1997; 6: 1248-1263Crossref PubMed Scopus (147) Google Scholar). For the1H/2D exchange studies, the 1H NMR sample was diluted by a factor of 2 in a deuterated phosphate buffer at 20 °C. A series of 15N HSQC spectra was acquired every 10 (four times), 40 (12 times), 80 (six times), and 160 (nine times) min. To identify the slowest exchangeable protons, a similar study was performed after a quick dialysis (2 h) against deuterated buffer. Three classes of exchange were defined: highly exchangeable protons (within 20 min), moderately protected (time of exchange 1 day). For the thermal studies, a series of 15N HSQC spectra was acquired at 50 μm protein concentration at different temperatures ranging from 5 °C to 50 °C in steps of 5 °C. All data were processed on a Silicon Graphics O2 work station with the program SNARF, version 0.8.9 (Frans van Hoesel, University of Groningen). For the titration experiments, a sample of the wild-type p13suc1 was prepared in a 50 mm Tris buffer as described previously (8.Landrieu I. Odaert B. Wieruszeski J.-M. Drobecq H. Inzé D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Increasing amounts of unlabeled synthetic peptide of sequence EQPLpTPVTDL (where pT is phosphothreonine) (phospho-Cdc25 peptide) were added to a 0.50 mm15N-labeled p13suc1sample. Final concentrations were successively 0.5/0.125 mm, 0.5/0.25 mm, 0.5/0.50 mm, 0.5/1.0 mm, 0.5/1.5 mm, 0.5/2.5 mm, 0.5/5.0 mm, and 0.5/10 mm.1H-15N HSQC spectra were measured at each titration point. The binding constant was calculated as described previously (8.Landrieu I. Odaert B. Wieruszeski J.-M. Drobecq H. Inzé D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The good dispersion of the 15N HSQC spectrum indicates that the protein adopts a folded conformation in solution, justifying further efforts to the determination of the solution conformation (Fig. 1). We therefore attempted to assign the backbone resonances by heteronuclear NMR experiments. A complete backbone assignment, with the exception of the single residue Arg-39 in the anion-binding site, and the three first N-terminal residues was obtained. 2B. Odaert, I. Landrieu, K. Dijkstra, G. Schuurman-Wolters, P. Casteels, J.-M. Wieruszeski, D. Inzé, R. Scheek, and G. Lippens, manuscript submitted. A differential line broadening was observed for certain residues, especially those located near the β4-hinge region (see below). The 15N HSQC spectrum of the wild-type protein showed overall the same dispersion as the hinge mutant, suggesting that the mutation does not affect the overall structure. However, despite the use of an extensive set of triple resonance experiments, only a partial assignment was achieved with 12 of the expected 108 backbone amide bond cross-peaks missing in the15N HSQC spectrum (Table I). The missing resonances correspond to residues in the C-terminal β-sheet of the crystal structure (residues 84–97), some direct neighbors of this stretch on the β2-strand (residues 39 and 40), and residue 66 on a loop that is close in space according to the crystal structure (Fig. 2a). Line broadening was observed at the extremities of the C-terminal β-sheet (stretches 97–100 and 79–83), in the N-terminal β-sheet (stretches 27–31 and 38–43), and in the 63–68 segment interacting with the β3-strand and the β-hinge region. HSQC spectra at lower concentrations still showed this line broadening, excluding weak affinity dimerization as the dominant broadening mechanism (25.Korchuganov D.S. Nolde S.B. Reibarkh M.Y. Orekhov V.Y. Schulga A.A. Ermolyuk Y.S. Kirpichnikov M.P. Arseniev A.S. J. Am. Chem. Soc. 2001; 123: 2068-2069Crossref PubMed Scopus (29) Google Scholar, 26.Pfuhl M. Chen H.A. Kristensen S.M. Driscoll P.C. J. Biomol. NMR. 1999; 14: 307-320Crossref PubMed Scopus (45) Google Scholar). As the most severe line broadening was observed in the C-terminal β-sheet, we raised the hypothesis that the β-hinge region (88H XPEPH93), which was previously described to act as a loaded molecular spring (16.Schymkowitz J.W.H. Rousseau F. Irvine L.R. Itzhaki L.S. Structure. 2000; 8: 89-100Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 17.Schymkowitz J.W.H. Rousseau F. Itzhaki L.S. J. Mol. Biol. 2000; 301: 199-204Crossref PubMed Scopus (16) Google Scholar, 21.Schymkowitz J.W.H. Rousseau F. Wilkinson H.R. Friedler A. Itzhaki L.S. Nat. Struct. Biol. 2001; 8: 888-892Crossref PubMed Scopus (50) Google Scholar), could be involved in a conformational exchange at the intermediate time scale. Small chemical shift perturbations were observed between both samples for the residues in the neighboring segment 61–67, potentially indicating subtle differences in local structure between the wild-type protein and its hinge mutant.Table IResidues whose correlation peak is missing in the HSQC spectrum of the wild type p13suc1 protein but is visible in the spectrum of the P90A mutantResidue numberResidue typeSecondary structureBinding site39Argβ2Phosphate66LeuLoop84Metβ385TyrHinge86GluHinge87ValHinge88HisHinge89ValHinge91GluHinge93HisHinge94Ileβ495Leuβ496Leuβ497Pheβ4Residue type and location in the different secondary structure elements are indicated. Open table in a new tab Residue type and location in the different secondary structure elements are indicated. Similarly to our previous interaction study involving the hinge mutant P90A (8.Landrieu I. Odaert B. Wieruszeski J.-M. Drobecq H. Inzé D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), we titrated a synthetic phosphorylated peptide of Cdc25 into a 15N-labeled p13suc1 sample and observed chemical shift changes in the corresponding HSQC spectra. The identified residues corresponded to those found for the mutant protein, confirming for both proteins a very similar binding pocket composed of residues Arg-30, Arg-39, Gln-78, Trp-82, and Arg-99 (Fig. 2b). However, when we performed a titration experiment with increasing amounts of phosphopeptide to determine an affinity constant, we found a value of 180 ± 20 μm, indicative of a 5-fold stronger binding of the phosphopeptide to the wild-type protein than to the hinge mutant (8.Landrieu I. Odaert B. Wieruszeski J.-M. Drobecq H. Inzé D. Lippens G. J. Biol. Chem. 2001; 276: 1434-1438Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Even at the highest concentration of peptide, however, no additional peaks corresponding to those only present in the HSQC spectrum of the mutant protein could be observed. As the mutation allowed a complete assignment of the spectrum, we decided to first characterize the structure of the CKS protein in solution on the basis of the mutant NMR data. The secondary structure derived on the basis of the 13Cα, 13Cβ, 13CO, and 1Hα chemical shift index CSI (27.Wishart D.S. Sykes B.D. J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1909) Google Scholar) was confirmed by characteristic sequential NOE contacts. NN(I, I + 1) and αN(I, I + 1) NOEs of, respectively, strong and weak intensity coincide with upfield shifts of the 1Hα/13Cα/13Co profile and downfield shifts of the 13Cβ frequencies in the segments 12–19, 45–49, 67–71, and 103–107. The three first segments correspond in the crystal structure, respectively, to the α1-, α2-, and α4-helices. The fourth segment, unsolved in the crystal structures due to dynamic disorder, adopts a helical conformation as observed for the CKS1 protein (14.Bourne Y. Watson M.H. Arvai A.S. Bernstein S.L. Reed S.I. Tainer J.A. Struct. Fold. Des. 2000; 8: 841-850Abstract Full Text Full Text PDF Scopus (35) Google Scholar). Despite the lack of helical CSI pattern, the segment 53–56 probably does adopt the short 310 α3-helix observed in the crystal structure as suggested by strong sequential NN(I, I + 1), αN(52, 54), and αN(53, 55) NOE contacts. Strong αN(I, I + 1) NOEs, associated with a significant downfield shift of the1Hα/13Cα/13Co profile and upfield 13Cβ values defined a β-extended conformation for the strands β1 (residues 26–28), β2 (residues 36–43), and β4 (residues 94–99) of the four-stranded β-sheet. The segments (8–11 and 78–80) are equally extended but not involved in the formation of β-sheet, and the absence of any secondary CSI pattern between residues 82 and 85 confirmed that the strand β3 is not in a regular conformation in solution in agreement with the crystal structure. The C-terminal elongation of the α1-helix and the β1-strand is interrupted by the presence of the proline residues Pro-20 and Pro-29 as supported by the disruption of the CSI pattern in the vicinity of these residues. The presence of nonsequential backbone αα, αN, and NN connectivities supports the presence of a four-stranded β-sheet in the solution structure (Fig. 3). The numerous nonsequential NOEs between the β2- and β4-strands indicate that they participate in the formation of a regular antiparallel β-sheet. Finally the reversal of the protein chain in the β-hinge region of the C-terminal β-sheet is supported by strong sequential NN(89, 90), NN(90, 91), and NN(93, 94) NOEs and weak intensity αN(89, 91), αβ(89, 92), and βN(89, 92) NOEs. All these results support the view that the protein adopts in solution the overall global fold of the closed form of the zinc-mediated dimer described by x-ray studies (11.Endicott J.A. Noble M.E. Garman E.F. Brown N. Rasmussen B. Nurse P. Johnson L.N. EMBO J. 1995; 14: 1004-1014Crossref PubMed Scopus (67) Google Scholar). 1H/2D exchange experiments were used to detail the protection of the amide protons against the solvent in terms of accessibility and hydrogen bonding. As our working pH for the NMR experiments was relatively high (>6.3), we observed that most of the amide protons in the mutant protein exchanged within the first 20 min. The remaining 36 residues showed intermediate exchange rates with a time constant around 24 h or very slow exchange rates with a protection extending over 2 days. Mapping of the three exchange rate classes of the P90A mutant protein on the crystal structure (Fig. 2c) shows that most of the highly protected residues are located in the β2-strand (residues 37, 38, 41, 42, and 43) and β4-strand (residues 94–99). In contrast to the central β-strands of the β-sheet, moderate protection was observed for the external β1-strand (residue 26) and β3-strand (residues 82 and 83). A similar trend was observed for the residues of the α1- (residues 15, 18, and 22), α2- (residues 47, 51), and α4-helices (residues 70, 71, 72) and hydrophobic residues 55, 66, 74, and 76 implied in the formation of the core of the protein. An exchange experiment on the wild-type protein gave very similar results, indicating that both proteins are protected to the same extent and in the same regions. Especially protection of amide protons of residues 37, 41, 43, 98, and 99 showed that the β4-strand is in a regular conformation in the wild-type protein as well (Fig. 2c). For a better characterization of the line broadening observed for the resonances of the four-stranded β-sheet in the wild-type and mutant proteins, we studied the effect of temperature on the NMR parameters by acquiring a series of 15N HSQC spectra for both proteins between 5 °C and 50 °C (Fig. 4). To prevent aggregation, the thermal study was performed at low protein concentration (50 μm) and at temperatures well below the unfolding temperature (65 °C). Raising the temperature led to a line narrowing for the resonances of most of the residues, probably due to an accelerated overall tumbling of the molecules. In contrast, resonances of the residues at the N-terminal and C-terminal extremities disappeared at higher temperatures due to an increase of their exchange rates with water. The major difference between the two proteins resides in the behavior of those residues whose resonances were broadened or missing from the spectrum of the wild-type protein. An increase of temperature made those resonances reappear at the same chemical shift values as the corresponding resonances in the mutant protein at the same temperature. In particular, residues in the β4-strand (from 93 to 97) and in the neighboring region of the β3-strand (from 65 to 67) showed this behavior (Fig. 4). Residues in the β-hinge region (from Tyr-85 to Glu-91) could not be identified in the wild-type protein spectrum even at high temperatures. These thermal studies also allowed us to determine the amide thermal coefficients. High values in disordered or accessible parts of the protein are usually indicative of a significant degree of flexibility. We observed that some of the residues in the β-hinge region of the mutant protein, such as His-88, Val-89, and His-93, displayed even higher coefficients than those found for the flexible N- and C-terminal extremities, suggesting a high degree of internal flexibility for the β-hinge region. Such results are in accordance with the conformational heterogeneity observed in the crystal structures for this region. A similar trend is observed for the residue Tyr-27 interacting with the proline Pro-29, which introduces a bulge in the β1-strand. For the rest of the protein, no significant difference in thermal coefficients between the wild-type and the mutant proteins was observed. The small CKS protein is both functionally and structurally a remarkable protein module. A ubiquitous subunit of the CDK·cyclin complexes, it is essential for a correct progression of the cell cycle (3.Brizuela L. Draetta G. Beach D. EMBO J. 1987; 6: 3507-3514Crossref PubMed Scopus (259) Google Scholar, 4.Patra D. Dunphy W.G. Genes Dev. 1996; 10: 1503-1515Crossref PubMed Scopus (81) Google Scholar, 28.Moreno S. Hayles J. Nurse P. Cell. 1989; 58: 361-372Abstract Full Text PDF PubMed Scopus (401) Google Scholar), but its precise role as a targeting unit with a clear definition of its multiple binding partners is still being investigated. As for its structure, x-ray crystallography has identified two distinct conformations, each associated with a different assembly state: the monomer, in which the β4-
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