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).

Although enzymes responsible for the cyclization of amino-terminal glutamine residues are present in both plant and mammal species, none have yet been characterized in bacteria. Based on low sequence homologies to plant glutaminyl cyclases (QCs), we cloned the coding sequences of putative microbial QCs from Zymomonas mobilis (ZmQC) and Myxococcus xanthus (MxQC). The two recombinant enzymes exhibited distinct QC activity, with specificity constants k(cat)/K(m) of 1.47±0.33 mm⁻¹ s⁻¹ (ZmQC) and 142±32.7 mm⁻¹ s⁻¹ (MxQC) towards the fluorescent substrate glutamine-7-amino-4-methyl-coumarine. The measured pH-rate profile of the second order rate constant displayed an interesting deviation towards the acidic limb of the pH chart in the case of ZmQC, whereas MxQC showed maximum activity in the mild alkaline pH range. Analysis of the enzyme variants ZmQCGlu⁴⁶Gln and MxQCGln⁴⁶Glu show that the exchanged residues play a significant role in the pH behaviour of the respective enzymes. In addition, we determined the three dimensional crystal structures of both enzymes. The tertiary structure is defined by a five-bladed β-propeller anchored by a core cation. The structures corroborate the putative location of the active site and confirm the proposed relation between bacterial and plant glutaminyl cyclases.

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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