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Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21

Abstract

Traditionally, well-defined three-dimensional structure has been thought to be essential for protein function. However, myriad biological functions are performed by highly dynamic, intrinsically disordered proteins (IDPs). IDPs often fold upon binding their biological targets and frequently show 'binding diversity' by targeting multiple ligands. We sought to understand the physical basis of IDP binding diversity and report here that the cyclin-dependent kinase (Cdk) inhibitor p21Cip1 adaptively binds to and inhibits the various Cdk–cyclin complexes that regulate eukaryotic cell division. Using results from NMR spectroscopy and biochemical and cellular assays, we show that structural adaptability of a helical subdomain within p21, termed LH, enables two other subdomains, D1 and D2, to specifically bind conserved surface features of the cyclin and Cdk subunits, respectively, within otherwise structurally distinct Cdk–cyclin complexes. Adaptive folding upon binding is likely to mediate the diverse biological functions of the thousands of IDPs present in eukaryotes.

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Figure 1: p21 and p27 adopt similar secondary structure when bound to Cdk2–cyclin A.
Figure 2: The LH subdomains of p21-KID and p27-KID show flexibility and disorder within ternary complexes with Cdk2–cyclin A.
Figure 3: The Cdk2–cyclin A–bound structures of subdomains D1 and D2 within the p21 constructs are unaffected by elongation or truncation of subdomain LH by three residues.
Figure 4: Elongation or truncation of subdomain LH within p21 significantly influences cell cycle regulation and interactions with Cdk–cyclin complexes.
Figure 5: Adaptive folding mediates promiscuous binding of p21 to the diverse Cdk–cyclin complexes that regulate cell division.

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Acknowledgements

The authors gratefully acknowledge S.I. Reed (Scripps Research Institute) for providing a vector to express a cyclin D1–Cdk4 fusion protein, L. Hengst (Innsbruck Medical University) for providing baculoviruses that coexpress Cdk6 and cyclin D1, B. Schulman (St. Jude Children's Research Hospital) and P.D. Adams (Fox Chase Cancer Center) for providing Rb constructs, C.J. Sherr (St. Jude Children's Research Hospital) for providing anti-sera for Cdk2 and Cdk4 and for stimulating discussions, M. Assem (St. Jude Children's Research Hospital) for technical assistance with cell cycle assays, C.-G. Park (St. Jude Children's Research Hospital) for preparation of Cdk–cyclin complexes for kinase assays, N. Pytel (St. Jude Children's Research Hospital) for preparation of p21-KID protein samples, E. Tuomanen (St. Jude Children's Research Hospital) for assistance with kinase assays and R. Ashmun (St. Jude Children's Research Hospital) for FACS analysis. The authors acknowledge support from US National Institutes of Health core grant P30CA21765 (St. Jude Children's Research Hospital), 5R01CA082491 (to R.W.K.), R01CA71907 (to M.F.R.), the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital and a US National Science Foundation CAREER Award (NSF MCB 0952514 to J.C.).

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R.W.K. and M.F.R. designed the research; Y.W., J.C.F., L.O., S.O., R.M. and J.C. performed the research; Y.W., J.C.F., L.O., S.O., J.C., M.F.R. and R.W.K. analyzed data; L.X. and J.S. provided critical technical assistance; and Y.W., J.C.F., L.O., S.O., J.C., M.F.R. and R.W.K. wrote the manuscript.

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Correspondence to Richard W Kriwacki.

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Wang, Y., Fisher, J., Mathew, R. et al. Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21. Nat Chem Biol 7, 214–221 (2011). https://doi.org/10.1038/nchembio.536

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