ABSTRACT
Drosophila Toll receptors are involved in embryonic development and the immune response of adult flies. In both processes, the only known Toll receptor ligand is the human nerve growth factor-like cystine knot protein Spätzle. Here we present the crystal structure of a 1:1 (nonsignaling) complex of the full-length Toll receptor ectodomain (ECD) with the Spätzle cystine knot domain dimer. The ECD is divided into two leucine-rich repeat (LRR) domains, each of which is capped by cysteine-rich domains. Spätzle binds to the concave surface of the membrane-distal LRR domain, in contrast to the flanking ligand interactions observed for mammalian Toll-like receptors, with asymmetric contributions from each Spätzle protomer. The structure allows rationalization of existing genetic and biochemical data and provides a framework for targeting the immune systems of insects of economic importance, as well as a variety of invertebrate disease vectors.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/immunology , Immunity, Innate , Signal Transduction , Toll-Like Receptors/metabolism , Animals , Drosophila Proteins/chemistry , Humans , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Toll-Like Receptors/chemistryABSTRACT
Drosophila Toll receptors are involved in embryonic development and in the immune response of adult flies. In both processes, the Toll receptor ligand is the NGF-like cystine knot protein Spätzle. Here we present the expression of Toll receptor ectodomain in Schneider cells at high yields and demonstrate a high affinity interaction with the refolded and trypsin-processed Spätzle cystine knot domain dimer. Poorly and anisotropically diffracting crystals of the complex could be improved by deglycosylation and dehydration, paving the way for structural analyses of the Toll-Spätzle interaction.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Toll-Like Receptors/metabolism , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , Crystallization , Drosophila/chemistry , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Gene Expression , Molecular Sequence Data , Protein Multimerization , Protein Refolding , Protein Structure, Tertiary , Toll-Like Receptors/chemistry , Toll-Like Receptors/geneticsABSTRACT
Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.
