Sequential Unfolding of Ankyrin Repeats in Tumor Suppressor p16

The ANK repeat is a ubiquitous 33-residue motif that adopts a β hairpin helix-loop-helix fold. Multiple tandem repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly by short-range interactions between residues close in sequence. The tumor suppressor p16 INK4 consists of four repeats and represents the minimal ANK folding unit. We found from Φ value analysis that p16 unfolded sequentially. The two N-terminal ANK repeats, which are distorted from the canonical ANK structure in all INK4 proteins and which are important for functional specificity, were mainly unstructured in the rate-limiting transition state for folding/unfolding, while the two C-terminal repeats were fully formed. A sequential unfolding mechanism could have implications for the cellular fate of wild-type and cancer-associated mutant p16 proteins.