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The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53 tumor suppressor-dependent cell growth control in humans and other organisms, mediates G1/S-phase arrest through inhibition of cyclin-dependent kinases (Cdks). The enzymatic activity of these kinases is essential for progress through the cell division cycle and one level of cell cycle regulation is exerted through inhibition of Cdks by a family of small proteins, including p21. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2-terminus. Using proteolytic mapping, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, HPLC and size-exclusion chromatography, we show that p21, active as a Cdk inhibitor, exists in an extended, non-globular conformation in the absence of its biological target and that p21 lacks the hallmarks of stable secondary and tertiary structure. We have developed an efficient approach to obtain detailed proteolytic maps that takes advantage of the high accuracy and sensitivity of MALDI mass spectrometry. Our method allows a proteolytic map to be obtained from a single mass spectrum for fragments produced from a single proteolytic reaction.

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.