Docking of Human Interleukin-15 to its Specific Receptor a Chain: Correlation Between Molecular Modeling and Mutagenesis Experimental Data Agne `s Que ´me ´ ner, 1,2 Je ´ro ˆ me Bernard, 1,2 Erwan Mortier, 1,2 Ariane Plet, 1,2 Yannick Jacques, 1,2 * and Vinh Tran 3 1 INSERM, U601, Groupe de Recherche Cytokines et Re ´cepteurs, Institut de Biologie, Nantes, France 2 Universite ´ de Nantes, UFR Me ´decine, IFR26, Institut de Biologie, Nantes, France 3 CNRS, UMR 6204, U3B Unite ´ de Biotechnologie, Biocatalyse et Biore ´gulation, Faculte ´ des Sciences et Techniques, Nantes, France ABSTRACT A structural model of the sushi domain of IL-15Ra was first obtained by homology modeling to study its interactions with IL-15 by means of molecular modeling, peptide scanning, and site-directed mutagenesis. From these experi- mental data, a putative interacting surface of IL- 15Ra with a previously published IL-15 model was inferred: Leu25, Leu44, and Glu46 of IL-15 and Arg35 of IL-15Ra were found to be key interfacial residues and were subsequently used as filters for the construction of docking solutions. Human IL-15/IL-15Ra complexes were constructed in two stages, with a preliminary docking procedure, treating the two partners as rigid bodies and using these filters. In this first stage, two classes of docking solutions were characterized. From a topological point of view, each solution could be derived from the other by reverse orientation of one partner in relation to the other. In a second stage, several further energy refinements clearly favored one solution. Moreover, this unique dock- ing solution was confirmed by molecular model- ing of IL-15 mutants previously built and tested in our laboratory. Finally, this complex model, which is a useful tool to study the IL-15/IL-15Ra interface, was topologically compared to IL-2/IL- 2Ra complexes (previous model in the literature and recent crystal structure). Proteins 2006;65: 623–636. V V C 2006 Wiley-Liss, Inc. Key words: IL-15; cytokine; receptor; protein–protein interactions; site-directed mutagenesis INTRODUCTION IL-15 is a pleiotropic cytokine that plays important roles in the innate and adaptive immune systems, including the development, activation, homing and sur- vival of immune effectors, especially NK, NK-T, and CD8 þ T cells. 1 IL-15 was initially identified by its ability to mimic in vitro IL-2-induced T-cell proliferation. 2 Both cytokines are structurally related, belonging to the four a-helix bundle family 3 and have similar in vitro biologi- cal properties, consistent with common receptor (R) sig- naling components (IL-2Rb and g c ). 2,4,5 The IL-2Rb/g c complex is a common receptor for IL-2 and IL-15 (with intermediate affinity: K d ¼ 1nM) and both cytokines compete with each other to bind to this complex. IL-2 and IL-15 both signal through this receptor and thus trigger similar downstream signaling pathways follow- ing activation of Jak-1 and Jak-3 tyrosine kinases. 6,7 Cytokine specificity is conferred by unique and specific a-chain receptors 8 that complete the IL-15 or IL-2 het- erotrimeric (abg) high-affinity (K d ¼ 0.05–0.1 nM) recep- tor complexes. 9–11 IL-2Ra- and IL-15Ra-chains are struc- turally related and present sequence homologies with b- sandwich domains previously found in some complement and adhesion molecules. 12 These structural domains called ‘‘sushi domains’’ (around 65 amino acids) are also known as SCRs, or CCP repeats. The IL-2Ra chain con- tains two such domains, both of which are involved in the IL-2/IL-2Ra interaction, although the N-terminal do- main bears most of the residues in contact with IL-2. 13 On the contrary, the IL-15Ra chain contains only one Abbreviations: ABTS, 2,2 0 -azino-di-3-ethylbenzthiazoline sulfonate; APC, antigen-presenting cell; CCD, charge-coupled device; CCP, com- plement control protein; DMEM, Dulbecco’s modified Eagle’s medium; EBNA, Epstein–Barr nuclear antigen; ELISA, enzyme- linked immunosorbent assay; FCS, fetal calf serum; GM-CSF, granu- locyte–macrophage colony stimulating factor; IC50, inhibitory con- centration 50%; IgG, immunoglobulin G; IL, interleukin; IL-15Ra, IL-15 receptor a chain; Jak, Janus kinase; mAb, monoclonal anti- body; NK, natural killer; PDB, Protein Data Bank; RIA, radioimmu- noassay; rIL, recombinant IL; SCR, short consensus repeat; WT, wild-type Grant sponsor: Institut National de la Recherche Me ´dicale (INSERM); Grant sponsor: Centre National de la Recherche Scienti- fique (CNRS); Grant sponsor: Association pour la Recherche sur le Cancer (ARC); Grant number: A03/1/3311; Grant sponsor: Ligue Nationale Contre le Cancer (LNCC; Comite ´ de Vende ´e) (fellowships to J. Bernard and E. Mortier); Grant sponsor: ARC (fellowships to J. Bernard and E. Mortier); Grant sponsor: Ministe `re de la Recher- che et des Technologies (fellowship to E. Mortier). *Correspondence to: Yannick Jacques, INSERM Unite ´ 601, De ´partement de Recherches en Cance ´rologie, 9 quai Moncousu, 44093 Nantes cedex 01, France. E-mail: yjacques@nantes.inserm.fr Received 9 December 2005; Revised 26 April 2006; Accepted 31 May 2006 Published online 25 September 2006 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21103 V V C 2006 WILEY-LISS, INC. PROTEINS: Structure, Function, and Bioinformatics 65:623–636 (2006)