Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways

Journal of Molecular Biology
Antoine DrevellePhilippe Minard

Abstract

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. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were o...Continue Reading

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Citations

Dec 16, 2011·Analytical and Bioanalytical Chemistry·Silvia Pavan, Federico Berti
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Dec 9, 2017·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Shun Hirota, Ying-Wu Lin
Apr 15, 2014·Dalton Transactions : an International Journal of Inorganic Chemistry·Elodie Sansiaume-DagoussetRémy Ricoux
May 6, 2009·Chembiochem : a European Journal of Chemical Biology·Antoine DrevellePhilippe Minard
Aug 28, 2020·Chembiochem : a European Journal of Chemical Biology·Wadih GhattasA Jalila Simaan

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