Tumor suppressor INK4: Quantitative structure-function analyses of p18(INK4C) as an inhibitor of cyclin-dependent kinase

We report the first detailed structure-function analyses of p18(INK4C) (p18 ), which is a homologue of the important tumor suppressor p16(INK4A) (p16). Twenty-four mutants were designed rationally. The global conformations of the mutants were characterized by NMR, while the function was assayed by in hibition of cyclin-dependent kinase 4 (CDK4). Most of these mutants have un perturbed global structures, thus the changes in their inhibitory abilities can be attributed to the mutated residues. The important results are summa rized as follows: (a) some residues at loops 1 and 2, but not 3, are import ant for the inhibitory function of p18, similar to the results for p16; (b) two residues at the first helix-turn-helix motif and two at the third are important for inhibition; (c) while the results generally agree with the pr ediction based on the crystal structures of p16-CDK6 and p19-CDK6 binary co mplexes, there are significant differences in a few residues, suggesting th at the interactions in the binary complexes may not accurately represent th e interactions in the ternary complexes tin the presence of cyclin D2); (d) most importantly, the extra loop of pls appears to contribute to the funct ion of p18, even though the crystal structure of the p19(INK4D)-CDK6 comple x indicates no interactions involving this loop; (e) detailed analyses of t he crystal structures and the functional results suggest that there are not able differences in the interactions between different members of the INK4 family and CDKs.