The Drosophila Spätzle protein, involved in the embryonic development of the dorsal-ventral axis and in the adult immune response, is expressed as a proprotein and is activated by the serine proteinases Easter or Spätzle-processing enzyme. Proteolytic cleavage generates a 106-amino acid COOH-terminal fragment, C106, homologous to the mature form of nerve growth factor NGF, a cystine knot protein. Through alternative splicing, the Spätzle gene encodes for several isoforms that (with one exception, the "propeptide isoform") share C106 but differ in the prosequence. Three isoforms have been expressed recombinantly in Escherichia coli strains. The propeptide isoform could be expressed in soluble form and is unstructured according to CD and NMR measurements. Dimeric full-length Spätzle isoforms have been refolded from insoluble inclusion bodies and are able to rescue Spätzle-deficient embryos. Although the two full-length isoforms exhibit similar far-UV CD spectra, large differences in tryptophan fluorescence quenching by the respective pro-parts are observed. Both full-length isoforms exhibited highly cooperative folding transitions. Proteolytic digestion using trypsin resulted in C106, whose unfolding exhibits lower thermodynamic stability and cooperativity compared with the full-length proteins. The structure of C106 reveals a T-shaped dimer with significant differences to NGF and a deep internal cavity. Substantial ␤-sheet formation is observed between the two monomers, whereas a long loop containing the single tryptophan residue is disordered in the crystals. Our results suggest that the propeptides stabilize the tertiary structure of the "mature" Spätzle cystine knot.The Spätzle protein is the precursor of a nerve growth factorlike ligand in Drosophila melanogaster (1). It defines the dorsalventral axis in Drosophila embryos and acts in the initiation of immune response to fungal and bacterial infection in adult flies (2). Sequence homology to coagulogen and human nerve growth factor (NGF), 2 together with the spacing of cysteine residues, suggests a cystine knot motif, in which two disulfide bridges form a ring through which a third disulfide bridge is threaded (3).Extracellular binding of mature Spätzle to its receptor Toll is thought to lead to receptor dimerization and autophosphorylation of the cytoplasmic Toll/interleukin-1 receptor domains (4, 5). Although Toll is distributed uniformly within the embryonic perivittelline membrane, a concentration gradient of Spätzle results in dorsal-ventral asymmetry. The extracellular domain of the transmembrane receptor Toll consists of two leucine-rich repeat domains that bind the ligand. The cytoplasmic domain of Toll shares sequence similarity with the vertebrate Toll-like receptors, such as interleukin-1 receptor (6, 7), also involved in innate immunity.Activation of Spätzle proceeds via one of two extracellular proteolytic cascades. In the developmental pathway, Spätzle is activated by Easter, generating a 12-kDa COOH-terminal fragment, C106, that is capable of a...

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