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. 1996 Oct 15;93(21):11504–11509. doi: 10.1073/pnas.93.21.11504

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

R W Kriwacki 1, L Hengst 1, L Tennant 1, S I Reed 1, P E Wright 1
PMCID: PMC38087  PMID: 8876165

Abstract

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chen J., Jackson P. K., Kirschner M. W., Dutta A. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature. 1995 Mar 23;374(6520):386–388. doi: 10.1038/374386a0. [DOI] [PubMed] [Google Scholar]
  2. Deng C., Zhang P., Harper J. W., Elledge S. J., Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell. 1995 Aug 25;82(4):675–684. doi: 10.1016/0092-8674(95)90039-x. [DOI] [PubMed] [Google Scholar]
  3. Dulić V., Kaufmann W. K., Wilson S. J., Tlsty T. D., Lees E., Harper J. W., Elledge S. J., Reed S. I. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell. 1994 Mar 25;76(6):1013–1023. doi: 10.1016/0092-8674(94)90379-4. [DOI] [PubMed] [Google Scholar]
  4. Fotedar R., Fitzgerald P., Rousselle T., Cannella D., Dorée M., Messier H., Fotedar A. p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity. Oncogene. 1996 May 16;12(10):2155–2164. [PubMed] [Google Scholar]
  5. Goubin F., Ducommun B. Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. Oncogene. 1995 Jun 15;10(12):2281–2287. [PubMed] [Google Scholar]
  6. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  7. Harper J. W., Elledge S. J., Keyomarsi K., Dynlacht B., Tsai L. H., Zhang P., Dobrowolski S., Bai C., Connell-Crowley L., Swindell E. Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell. 1995 Apr;6(4):387–400. doi: 10.1091/mbc.6.4.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hengst L., Dulic V., Slingerland J. M., Lees E., Reed S. I. A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5291–5295. doi: 10.1073/pnas.91.12.5291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lee M. H., Reynisdóttir I., Massagué J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev. 1995 Mar 15;9(6):639–649. doi: 10.1101/gad.9.6.639. [DOI] [PubMed] [Google Scholar]
  10. Logan T. M., Thériault Y., Fesik S. W. Structural characterization of the FK506 binding protein unfolded in urea and guanidine hydrochloride. J Mol Biol. 1994 Feb 18;236(2):637–648. doi: 10.1006/jmbi.1994.1173. [DOI] [PubMed] [Google Scholar]
  11. Luo Y., Hurwitz J., Massagué J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1. Nature. 1995 May 11;375(6527):159–161. doi: 10.1038/375159a0. [DOI] [PubMed] [Google Scholar]
  12. Macleod K. F., Sherry N., Hannon G., Beach D., Tokino T., Kinzler K., Vogelstein B., Jacks T. p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 1995 Apr 15;9(8):935–944. doi: 10.1101/gad.9.8.935. [DOI] [PubMed] [Google Scholar]
  13. Matsuoka S., Edwards M. C., Bai C., Parker S., Zhang P., Baldini A., Harper J. W., Elledge S. J. p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev. 1995 Mar 15;9(6):650–662. doi: 10.1101/gad.9.6.650. [DOI] [PubMed] [Google Scholar]
  14. Nakanishi M., Robetorye R. S., Adami G. R., Pereira-Smith O. M., Smith J. R. Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1. EMBO J. 1995 Feb 1;14(3):555–563. doi: 10.1002/j.1460-2075.1995.tb07031.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Neri D., Wider G., Wüthrich K. 1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea. FEBS Lett. 1992 Jun 1;303(2-3):129–135. doi: 10.1016/0014-5793(92)80504-a. [DOI] [PubMed] [Google Scholar]
  17. Noda A., Ning Y., Venable S. F., Pereira-Smith O. M., Smith J. R. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res. 1994 Mar;211(1):90–98. doi: 10.1006/excr.1994.1063. [DOI] [PubMed] [Google Scholar]
  18. Parker S. B., Eichele G., Zhang P., Rawls A., Sands A. T., Bradley A., Olson E. N., Harper J. W., Elledge S. J. p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science. 1995 Feb 17;267(5200):1024–1027. doi: 10.1126/science.7863329. [DOI] [PubMed] [Google Scholar]
  19. Polyak K., Lee M. H., Erdjument-Bromage H., Koff A., Roberts J. M., Tempst P., Massagué J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994 Jul 15;78(1):59–66. doi: 10.1016/0092-8674(94)90572-x. [DOI] [PubMed] [Google Scholar]
  20. Rosenblatt J., De Bondt H., Jancarik J., Morgan D. O., Kim S. H. Purification and crystallization of human cyclin-dependent kinase 2. J Mol Biol. 1993 Apr 20;230(4):1317–1319. doi: 10.1006/jmbi.1993.1248. [DOI] [PubMed] [Google Scholar]
  21. Russo A. A., Jeffrey P. D., Patten A. K., Massagué J., Pavletich N. P. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature. 1996 Jul 25;382(6589):325–331. doi: 10.1038/382325a0. [DOI] [PubMed] [Google Scholar]
  22. Shou W., Dunphy W. G. Cell cycle control by Xenopus p28Kix1, a developmentally regulated inhibitor of cyclin-dependent kinases. Mol Biol Cell. 1996 Mar;7(3):457–469. doi: 10.1091/mbc.7.3.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Spolar R. S., Record M. T., Jr Coupling of local folding to site-specific binding of proteins to DNA. Science. 1994 Feb 11;263(5148):777–784. doi: 10.1126/science.8303294. [DOI] [PubMed] [Google Scholar]
  24. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  25. Su J. Y., Rempel R. E., Erikson E., Maller J. L. Cloning and characterization of the Xenopus cyclin-dependent kinase inhibitor p27XIC1. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10187–10191. doi: 10.1073/pnas.92.22.10187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toyoshima H., Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994 Jul 15;78(1):67–74. doi: 10.1016/0092-8674(94)90573-8. [DOI] [PubMed] [Google Scholar]
  27. Waga S., Hannon G. J., Beach D., Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994 Jun 16;369(6481):574–578. doi: 10.1038/369574a0. [DOI] [PubMed] [Google Scholar]
  28. Zhang O., Kay L. E., Olivier J. P., Forman-Kay J. D. Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques. J Biomol NMR. 1994 Nov;4(6):845–858. doi: 10.1007/BF00398413. [DOI] [PubMed] [Google Scholar]
  29. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

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