The effect of tumor-derived mutations and the role of ankyrin repeats on stability of p16: A paradigm for structure-function studies

p16INK4 (thereafter p16) is a specific cyclin D-dependent kinase inhibitor that regulates the G1 phase of cell cycle progression. Inactivation of p16 is the second most common genetic alteration in human cancer. In particular, many loss of function mutations in p16 are well-documented to associate with familial melanoma and other inherited malignant diseases. ^ To understand the effect of tumor-derived mutations on the conformational stability of p16, we first investigated the structure and oligomerization state of seven loss of function p16 mutant proteins. Our results showed that these proteins are either globally unfolded or assume a partially folded conformation. Some of these proteins are highly prone to aggregation. The location of the unfolded regions in two mutant p16 proteins was further investigated by limited proteolysis. In both proteins, the second ankyrin (ANK) repeat region and the β-hairpin loops connecting this repeat with the rest of the protein are significantly unfolded. These regions are important for cyclin dependent kinase binding. Thus, we propose that the unfolding of the second ANK repeat region and intermolecular aggregation could be a common mechanism for inactivation of p16. ^ p16 contains four ANK repeats, a 33-residue sequence motif that mediates protein-protein interactions. To understand the role of individual ANK repeats on stability of p16, we have constructed a subdomain, called p16C, which encompasses the third and fourth ANK repeats of p16. p16C has moderately stable secondary and tertiary structure, with the ANK repeats adopting a similar topological fold as in the full-length protein. Further dissection of p16C into two complementary peptides, each containing a single ANK repeat, results in molecules that are unstructured in solution. Thus, p16C is the smallest ANK repeat module that can fold independently and the two ANK repeat fold may represent the minimum structural unit for all ANK repeat proteins. ^ In summary, p16 is a protein with low thermodynamic stability. Many tumor-derived mutations disrupt the structure of p16, thus inactivating its function. The stability of p16 is primarily determined by the C-terminal two ANK repeats. Future studies will provide additional insights into the structure-function relationships of this important tumor suppressor protein.