An ancient reaction vessel: TobZ carbamoylates the antibiotic tobramycin to form nebramycin 5′. The YrdC‐like domain (blue) catalyzes the formation of the novel intermediate carbamoyladenylate, which is channeled through a common “reaction chamber” to the Kae1‐like domain (brown), site of carbamoyl transfer.

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.Toll signaling | embryonic morphogenesis | protein evolution T he model organism Drosophila melanogaster has yielded valuable insights into a number of fundamental biological processes. Genetic screens of flies subjected to chemical saturation mutagenesis have revealed a host of genes necessary for development, including Toll (from the German for "fantastic"), identified as a major determinant in the development of dorsalventral polarity (1-3). Toll encodes for a type I integral membrane protein with a large N-terminal extracellular domain consisting of a series of leucine-rich repeats (LRRs) (4) flanked by cysteine-rich motifs (5). The cytoplasmic intracellular C-terminal domain shares significant similarities with the mammalian interleukin-1 receptor (6, 7) and thus is termed the Toll-interleukin receptor (TIR) domain.Microinjection experiments have identified the spz gene product Spätzle as the ligand for Toll (8, 9). The morphogen, present throughout the perivitelline space in the form of an inactive precursor proSpätzle, undergoes activation cleavage by the serine protease Easter (10) (itself activated during development by a spatially confined proteolytic cascade), leading to an extracellular gradient of activated Spätzle in the developing embryo (reviewed in ref. 11). Spätzle-mediated activation of the Toll receptor results in nuclear localization of the NF-κB/rel transcription factor Dorsal (12), eliciting transcription of further ventral-specific differentiation genes. In addition, the absence of nuclear Dorsal protein on the dorsal side of the embryo derepresses another distinct class of genes (13).

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.

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.

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