Structures of in Vitro Evolved Binding Sites on Neocarzinostatin Scaffold Reveal Unanticipated Evolutionary Pathways
| Autor: | Marc Graille, Bernadette Heyd, Nathalie Ulryck, Isabelle Sorel, Antoine Drevelle, Herman van Tilbeurgh, Philippe Minard, Frédéric Pecorari, Michel Desmadril |
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| Přispěvatelé: | Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular Phage display Protein Conformation Stereochemistry Neocarzinostatin [SDV]Life Sciences [q-bio] Mutant Sequence (biology) In Vitro Techniques Biology Crystallography X-Ray Ligands 010402 general chemistry 01 natural sciences Evolution Molecular 03 medical and health sciences Zinostatin Structural Biology medicine Testosterone structure Binding site directed evolution Molecular Biology 030304 developmental biology 0303 health sciences Binding Sites binding site Directed evolution Ligand (biochemistry) 0104 chemical sciences phage display Systematic evolution of ligands by exponential enrichment Protein Binding medicine.drug |
| Zdroj: | Journal of Molecular Biology Journal of Molecular Biology, Elsevier, 2006, 358, pp.455-471. ⟨10.1016/j.jmb.2006.02.002⟩ |
| ISSN: | 0022-2836 1089-8638 |
| DOI: | 10.1016/j.jmb.2006.02.002⟩ |
| Popis: | International audience; 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 sidechains. 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 observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments. |
| Databáze: | OpenAIRE |
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