FC0086
p21^Cip1 cyclin-dependent kinase inhibitor (p21^WAF1/CIP1)  -  Cdk2/cyclin

Biological function
p21 is a universal inhibitor of the Cdk/cyclin repertoire (Cdk1, Cdk2, Cdk4 and Cdk6 paired with their respective cyclin partners e.g., cyclin A, B1, B2, D1 and D3) and is an important regulator of the cell cycle. p21 has been shown to activate Cdk4 (and Cdk6). p21-dependent inhibition of cell cycle progression from G₁ to S phase is mediated by inhibition of Cdk4/(and Cdk6)/D-type cyclin complexes, and Cdk2/cyclin E (and cyclin A) complexes. Additionally, p21-dependent arrest in G₂ phase is mediated by inhibition of Cdk1/cyclin B1.

Domain organization/sequence features
The N-terminal KID of p21 (residues 17–78; p21-KID) can be divided into three sub-domains: D1 (17-26), LH (27-48) and D2 (49-78). D1 of p21 binds to the cyclin subunit of Cdk/cyclin complexes and sub-domain D2 binds to the Cdk subunit.

Structural evidence
Sub-domain LH, which adopts a partially α-helical conformation, plays primarily a structural role by tethering sub-domains D1 and D2. Amides at the extreme C-terminus of sub-domain LH, appeared to be rigid. In contrast, amides in the center of this sub- domain (residues 34–44) exhibit hetNOE values between +0.3 and +0.4, indicative of increased mobility in comparison with those in sub-domains D1 and D2. Further, resonances for seven residues within the N-terminal portion of sub-domain LH were not observed, possibly due to dynamic conformational exchange. Thus hetNOE results suggested that many residues within sub-domain LH remain dynamic when p21-KID was bound to Cdk2/cyclin A. Multidisciplinary studies revealed that, when p21 is bound to Cdk2/cyclin A, sub-domain LH is not rigid but rather dynamic, allowing it to serve as an adaptable linker between sub-domains D1 and D2.

Biochemical evidence
p21-KID fully inhibits Cdk2/cyclin A with a K_i value of 0.3 nM, indicative of thermodynamically very favorable interactions between p21-KID and Cdk2/cyclin A. The presence of sub-domains D1 and D2, connected by the LH sub- domain, is associated with high Cdk2 inhibitory potency (IC₅₀ < 5 nM), despite the relatively weak binding of p21-D2 to Cdk2. p21-KID exhibited similar IC₅₀ values toward Cdk4/cyclin D1 and Cdk6/cyclin D1 (7.5 and 11 nM, respectively). Variants, where LH was increased or decreased by three residues (p21-KID-LH₊₃ and p21- KID-LH₋₃) were significantly less potent toward these complexes, despite the very similar structural characteristics. Thus LH dynamics affects p21 promiscuity. p21 LH sub-domain variants were also variably deficient in G₁/S and G₂ arrest.

Structure/Mechanism
The p21-KID-LH₊₃ ternary complex was slightly more stable than the complex with wild-type p21-KID and that the ternary complex that contained p21-KID-LH_-3 was significantly less stable, which is consistent with the degree of stretching of the LH sub-domain. The LH of p21-KID-LH₊₃ may be stretched to the smallest extent relative to a standard α-helix, while that of p21-KID-LH_-3 may be stretched to the greatest extent.

Mechanism category
conformational selection/tethering

Significance
Fuzziness of sub-domain LH allows p21 to adaptively bind to the individual complexes which comprise the Cdk/cyclin repertoire that regulates cell division.