Structures of in Vitro Evolved Binding Sites on Neocarzinostatin Scaffold Reveal Unanticipated Evolutionary Pathways

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways.