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Gankyrin is an ankyrin repeat protein known for its oncogenic effects and its up-regulation in the early stages of almost all cases of hepatocellular carcinoma, as well as other cancers. It mediates its oncogenic effects via a series of protein-protein interactions with several key regulators of the cell cycle including, but not limited to, pRb, CDK4 and Mdm2. A better understanding of these interactions, including their structural details, would facilitate the development of novel therapeutics targeting gankyrin, as well as improving our understanding of gankyrin and the cell cycle control mechanisms it alters. The nature of gankyrin's interaction with pRb is the subject of some dispute within the literature, including whether gankyrin is in a folded or unfolded state when interacting with pRb. Previous studies have identified that hydroxylation of ankyrin repeat proteins by FIH-1 requires substrate unfolding. Therefore, an investigation of the hydroxylation of gankyrin and other substrates demonstrates that gankyrin does not unfold when in complex with the C-terminal domain of the proteasomal S6 ATPase subunit. Molecular dynamics simulations of FIH-1 in complex with substrates derived from ankyrin repeats suggest that local sequence effects, as well as the tendency of the repeat to unfold, can determine whether or not an ankyrin repeat protein can be hydroxylated by FIH-1. Using hydroxylation to detect protein unfolding, this study provides further evidence that gankyrin interacts with pRb in a folded state, and via the use of in silico methods, proposes a structural model of the interaction. Mutations in gankyrin and pRb that are predicted to disrupt the …