Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30−34 amino acid residues and exclusively functions to mediate protein−protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix−turn−helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix−turn−helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.

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The solution structure of the tumor suppressor p16INK4A has been determined by NMR, and important recognition regions of both cdk4 and p16INK4A have been identified. The tertiary structure of p16INK4A contains four helix-turn-helix motifs linked by three loops. Twelve tumorigenic mutants of p16INK4A have been constructed and analyzed for their structure and activity, and new mutants have been designed rationally. A fragment of 58 residues at the N terminus of cdk4 important for p16INK4A binding has been identified. The importance of this region was further verified by mutational analysis of cdk4. These results and docking experiments have been used to assess possible modes of binding between p16INK4A and cdk4.

P16INK4A (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly due to the prevalence of genetic inactivation of the p16INK4A (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that elevated expression (up-regulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications in the structure-function relationship of P16 and in human cancers.