Evolutionary and functional epitopes of the Spätzle protein: New insights into activation of the Toll receptor

Sp?tzle, a dimeric ligand, binds to the Drosophila Toll receptor and activates the signal pathway functioning in both embryonic patterning and innate immunity. Here, we used the evolutionary trace approach based on phylogenetic information to predict the evolutionary epitope of Sp?tzle and found that it mainly clusters in several adjacent loops of Sp?tzle far from the cystine-knot structural domain. We designed six mutants of Sp?tzle based on the evolutionary epitope and transfected them into a stable cell line expressing the luciferase reporter gene under the control of the drosomycin promoter. Luciferase assays showed that these mutants cannot significantly activate the drosomycin promoter, suggesting the involvement of these sites in binding of Sp?tzle to the Toll receptor. These data highlight the importance of the Trp-loop of the mushroom-shaped Sp?tzle dimer in Toll receptor activation and demonstrate that evolution-guided site-specific mutagenesis represents a useful and promising strategy for understanding the ligand-receptor interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 14 January 2009; received after revision 14 February 2009; accepted 09 March 2009