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A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein–protein interaction involving the oncoprotein gankyrin. However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly. We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin–S6 ATPase binding interaction by isothermal titration calorimetry.

U1A binds U1hpII, a hairpin RNA with a 10-nucleotide loop. A U1A mutant (ΔK50ΔM51) binds U1hpII-derived hairpins with shorter loops, making it an interesting scaffold for engineering or evolving proteins that bind similarly sized disease-related hairpin RNAs. However, a more detailed understanding of complexes involving ΔK50ΔM51 is likely a prerequisite to generating such proteins. Toward this end, we measured mutational effects for complexes involving U1A ΔK50ΔM51 and U1hpII-derived hairpin RNAs with seven- or eight-nucleotide loops and identified contacts that are critical to the stabilization of these complexes. Our data provide valuable insight into sequence-selective recognition of seven- or eight-nucleotide loop hairpins by an engineered RNA binding protein.