Abstract
Overexpression of the ankyrin repeat oncoprotein gankyrin is directly linked to the onset, proliferation, and/or metastasis of many cancers. The role of gankyrin in multiple disease-relevant biochemical processes is profound. In addition to other cellular processes, gankyrin overexpression leads to decreased cellular levels of p53, through a complex that involves MDM2. Thus, inhibition of this interaction is an attractive strategy for modulating oncogenic phenotypes in gankyrin-overexpressing cells. However, the lack of well-defined, hydrophobic, small-molecule binding pockets on the putative ankyrin repeat binding face presents a challenge to traditional small-molecule drug discovery. In contrast, by virtue of their size and relatively high folding energies, synthetic gankyrin-binding proteins could, in principle, compete with physiologically relevant PPIs involving gankyrin. Previously, we showed that a shape-complementary protein scaffold can be resurfaced to bind gankyrin with moderate affinity (KD ∼6 μM). Here, we used yeast display high-throughput screening, error-prone PCR, DNA shuffling, and protein engineering to optimize this complex. The best of these proteins bind gankyrin with excellent affinity (KD ∼21 nM), selectively co-purifies with gankyrin from a complex cellular milieu, modulates an interaction between gankyrin and a physiological binding partner (S6 ATPase), and suppresses gankyrin/MDM2-dependent ubiquitination of p53.