title: Synthetic Proteins Potently and Selectively Bind the Oncoprotein Gankyrin, Modulate Its Interaction with S6 ATPase, and Suppress Gankyrin/MDM2-Dependent Ubiquitination of p53.
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.
authors.affiliation: †Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States.
authors.forename: Alex M
authors.initials: AM
authors.lastname: Chapman
authors.affiliation: †Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States.
authors.forename: Brian R
authors.initials: BR
authors.lastname: McNaughton
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D017089:Ankyrin Repeat; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000187:drug effects*; D011506:Proteins / Q000737:chemistry* / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: C403591:PSMD10 protein, human; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases