Structures of in Vitro Evolved Binding Sites on Neocarzinostatin Scaffold Reveal Unanticipated Evolutionary Pathways Antoine Drevelle 1 †, Marc Graille 2 †, Bernadette Heyd 1 , Isabelle Sorel 2 Nathalie Ulryck 2 , Fre ´de ´ ric Pecorari 1 , Michel Desmadril 1 , Herman van Tilbeurgh 2 and Philippe Minard 1 * 1 Equipe de Mode ´lisation et Inge ´nierie des Prote ´ines IBBMC, CNRS, UMR8619 Universite ´ Paris XI, Ba ˆt. 430 91405 Orsay, France 2 Equipe de Ge ´nomique Structurale de la Levure IBBMC, CNRS, UMR8619 Universite ´ Paris XI, Ba ˆt. 430 91405 Orsay, France 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 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 arrange- ment used for the selection experiments. q 2006 Elsevier Ltd. All rights reserved. Keywords: phage display; Neocarzinostatin; structure; binding site; directed evolution *Corresponding author Introduction Recent progress in combinatorial engineering of proteins has opened the possibility to introduce tailored binding sites in proteins. Efforts in this direction are mainly driven by the need for specific reagents for analytical as well as therapeutic applications. Although polyclonal or monoclonal antibodies are by far the most common source of specific reagents, alternative protein scaffolds are currently explored as a possible source of reagent with tailored specificity. 1–4 Combinatorial engin- eering relies on experimental processes that partially mimic the immune system: large libraries of protein variants have been created and selected by cell, 5,6 phage 7 or ribosome 8 display as well as by genetics methods. 9 These strategies allow us to bypass the constraints inherent to the immune system as well as to overcome the poor expression properties of antibodies, or antibody derivatives, 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. † A.D. and M.G. contributed equally to the work. Present addresses: B. Heyd, De ´partement d’Inge ´nierie et d’e ´tudes des Prote ´ines, Bat 152, CEA Saclay, Ba ˆt. 152, 91191 Gif-Sur-Yvette Cedex, France; F. Pecorari, Unite ´ de biochimie Structurale, CNRS, URA2185, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France. Abbreviations used: NCS, Neocarzinostatin; THS, testosterone hemisuccinate. E-mail address of the corresponding author: philippe.minard@ibbmc.u-psud.fr doi:10.1016/j.jmb.2006.02.002 J. Mol. Biol. (2006) 358, 455–471