Glycosciences Very Important Paper DOI: 10.1002/anie.201404568 Membrane Deformation by Neolectins with Engineered Glycolipid Binding Sites** Julie Arnaud, Kevin Trçndle, Julie Claudinon, Aymeric Audfray, Annabelle Varrot, Winfried Rçmer,* and Anne Imberty* Abstract: Lectins are glycan-binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glyco- lipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosy- lated surface and to invaginate glycolipid-containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites. By deciphering the glycocode, lectins play crucial roles in many biological or pathological processes. [1] Whereas indi- vidual lectin binding sites have rather low affinity for glycans, multivalency results in high avidity for carbohydrates pre- sented in several copies on glycoconjugates at the cell surface. [2] Moreover, multivalency also triggers the clustering of glycoproteins or glycolipids on cell surfaces. [3] Shiga and cholera toxin B-subunits as well as capsid lectins from SV40 and norovirus have been demonstrated to cluster glycosphin- golipids, resulting in negative membrane curvature and formation of membrane invaginations. [4] Multivalency of carbohydrates has been extensively studied, but not that of lectins, mainly because of the difficulty to control the number and location of binding sites in oligomeric proteins. An alternative approach is to use lectins presenting internal repeats such as b-propellers that are built on the cyclic repetition of 5 to 7 b-sheets. [5] The bacterial lectin, RSL from Ralstonia solanacearum, is an ancestor of this fold, as it consists of a tandem repeat of two b- sheets that trimerizes as a six-bladed b-propeller (Fig- ure 1 a,b). [6] The resulting hexavalent trimer has the capacity to invaginate lipid membranes containing fucosylated glyco- lipids. [7] Reducing the valency from six to three resulted in a trivalent lectin that lost the capacity to induce membrane invaginations in giant liposomes. [7] However, the influence of topology, that is, repartition of binding sites in space and the distance between them, has not been investigated. We therefore propose herein a novel approach for controlling valency. The “neolectin” concept consists in the Figure 1. a) Overall structure of wt-RSL complexed with aMeFuc dis- played as spheres (PDB code: 2BT9). Each monomer is represented by a different color. b) Representation of protein RSL and gene encoding RSL. The fucose binding sites are represented by white circle. c) Representation of protein neoRSL_VI and gene encoding neo- RSL_VI. [*] J. Arnaud, Dr. A. Audfray, Dr. A. Varrot, Dr. A. Imberty CERMAV, CNRS and Grenoble Alpes UniversitØ 38000 Grenoble (France) E-mail: imberty@cermav.cnrs.fr K. Trçndle, Dr. J. Claudinon, Prof. W. Rçmer Institute of Biology II, Albert-Ludwigs-University Freiburg Schänzlestrasse 1, 79104 Freiburg (Germany) and BIOSS Centre for Biological Signalling Studies Albert-Ludwigs-University Freiburg Schänzlestrasse 18, 79104 Freiburg (Germany) E-mail: winfried.roemer@bioss.uni-freiburg.de [**] The authors acknowledge funding from Agence Nationale de la Recherche Grant NeoLect (ANR-11-BSV5-002) (W.R., A.A. A.V., and A.I.), from CNRS (A.I. and A.V.), and from UniversitØ Grenoble Alpes (J.A.). W.R. is supported by the Excellence Initiative of the German Research Foundation (EXC 294), by a grant from the Ministry of Science, Research and the Arts of Baden-Württemberg (Az: 33-7532.20) and by a starting grant of the European Research Council (Programme “Ideas”—call identifier: ERC-2011-StG 282105). The COST actions CM1102 and BM1003 and the Labex ARCANE (ANR-11-LABX-003) are thanked for support. The authors are grateful to the SOLEIL for provision of synchrotron radiation facilities and to the Proxima 1 beam line staff for efficient assistance. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201404568. A ngewandte Chemi e 9267 Angew. Chem. Int. Ed. 2014, 53, 9267 –9270  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim