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Proc Natl Acad Sci U S A. Oct 15, 1996; 93(21): 11504–11509.
PMCID: PMC38087

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

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

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  • Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. [PubMed]
  • el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. [PubMed]
  • Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res. 1994 Mar;211(1):90–98. [PubMed]
  • Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell. 1995 Aug 25;82(4):675–684. [PubMed]
  • Parker SB, Eichele G, Zhang P, Rawls A, Sands AT, Bradley A, Olson EN, Harper JW, Elledge SJ. p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science. 1995 Feb 17;267(5200):1024–1027. [PubMed]
  • Macleod KF, 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. [PubMed]
  • Dulić V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell. 1994 Mar 25;76(6):1013–1023. [PubMed]
  • Waga S, Hannon GJ, 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. [PubMed]
  • Nakanishi M, Robetorye RS, Adami GR, Pereira-Smith OM, Smith JR. Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1. EMBO J. 1995 Feb 1;14(3):555–563. [PMC free article] [PubMed]
  • Chen J, Jackson PK, Kirschner MW, Dutta A. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA. Nature. 1995 Mar 23;374(6520):386–388. [PubMed]
  • 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. [PubMed]
  • Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, 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. [PubMed]
  • 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. [PubMed]
  • Su JY, Rempel RE, Erikson E, Maller JL. 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. [PMC free article] [PubMed]
  • Shou W, Dunphy WG. Cell cycle control by Xenopus p28Kix1, a developmentally regulated inhibitor of cyclin-dependent kinases. Mol Biol Cell. 1996 Mar;7(3):457–469. [PMC free article] [PubMed]
  • Lee MH, 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. [PubMed]
  • Matsuoka S, Edwards MC, Bai C, Parker S, Zhang P, Baldini A, Harper JW, Elledge SJ. 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. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Neidhardt FC, Bloch PL, Smith DF. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. [PMC free article] [PubMed]
  • Rosenblatt J, De Bondt H, Jancarik J, Morgan DO, Kim SH. Purification and crystallization of human cyclin-dependent kinase 2. J Mol Biol. 1993 Apr 20;230(4):1317–1319. [PubMed]
  • Hengst L, Dulic V, Slingerland JM, Lees E, Reed SI. A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5291–5295. [PMC free article] [PubMed]
  • Zhang O, Kay LE, Olivier JP, Forman-Kay JD. 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. [PubMed]
  • 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. [PubMed]
  • Logan TM, Thériault Y, Fesik SW. Structural characterization of the FK506 binding protein unfolded in urea and guanidine hydrochloride. J Mol Biol. 1994 Feb 18;236(2):637–648. [PubMed]
  • Spolar RS, Record MT., Jr Coupling of local folding to site-specific binding of proteins to DNA. Science. 1994 Feb 11;263(5148):777–784. [PubMed]
  • Harper JW, Elledge SJ, Keyomarsi K, Dynlacht B, Tsai LH, Zhang P, Dobrowolski S, Bai C, Connell-Crowley L, Swindell E, et al. Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell. 1995 Apr;6(4):387–400. [PMC free article] [PubMed]
  • Goubin F, Ducommun B. Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. Oncogene. 1995 Jun 15;10(12):2281–2287. [PubMed]
  • 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]
  • Russo AA, Jeffrey PD, Patten AK, Massagué J, Pavletich NP. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature. 1996 Jul 25;382(6589):325–331. [PubMed]

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