Residual structure within the disordered C-terminal segment of p21Waf1/Cip1/Sdi1 and its implications for molecular recognition

Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein-protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C-terminal segment of p21 encompassing residues 145-164 using a combination of NMR measurements and molecular dynamics simulations. The N-terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C-terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145-164) pre-exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets.