Designed Ankyrin Repeats as Enzyme inhibitors
Andreas Plueckthun, Patrick Amstutz, Kaspar Binz, Petra Parikez, Markus Gruetter

Repeat proteins are, next to antibodies, the most abundant classes of specific binding proteins in the genome. They can also work in the intracellular milieu, where antibodies are aggregation-prone, since disulfides cannot form. We have used consensus design to exploit the information in the sequence databases and design a library of ankyrin repeat proteins. Crystal structures suggest why the synthetic molecules surpass the natural repeat proteins in biophysical properties. Very rapid enrichment with ribosome display, an in vitro evolution method, against a variety of protein targets was observed, presumably due to their efficient folding. Binders against Maltose Binding Protein in the low nanomolar range have been obtained, and a crystal structure of the complex explains the antibody-like specificity. For a prokaryotic kinase and a protease, ankyrin-based inhibitors have been selected, and a crystal structure of both complexes explains their mode of action. This has for the first time allowed an "induced conformational change" in the target, with great potential for drug design. Structural biology will be helpful in the further engineering of repeat proteins, and repeat proteins will be useful tools in structural biology.

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