Abstract
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 beta-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.
Highlights
Our results suggest that the propeptides stabilize the tertiary structure of the “mature” Spatzle cystine knot
The Spatzle protein is the precursor of a nerve growth factorlike ligand in Drosophila melanogaster [1]
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]
Summary
Expression Vectors, Constructs, and Strains—Spatzle cDNAs, kindly provided by Dr Robert DeLotto, were amplified by polymerase chain reaction using the following primers: Spz11.7, 5Ј-GGC AAG CAT ATG AAG GAG TAC GAA CGT ATC ATC-3Ј (forward) and 5Ј-AAA CTC GAG CCC AGT CTT CAA CGC GCA CTT-3Ј (reverse); Spz8.19, 5Ј-GGC AAG CAT ATG AAG GAG TAC GAA CGT ATC ATC-3Ј (forward) and 5Ј-AAA GGATCC TCA CCC AGT CTT CAA CGC GCA CTT-3Ј (reverse); and Spz8.24, 5Ј-GGC AAG CAT ATG ACG TCC GCG GAC AGT GCA CCC-3Ј (forward) and 5Ј-AAT GGATCC TTA TTG ACA ATT AAC TGC CAG GTG TCG-3Ј (reverse). Elution was achieved using a 20 column volume gradient to 1 M NaCl. The final size exclusion step was carried out as for the full-length isoforms. The cystine knot domain with a COOHterminal His6-tag (C106His) was purified by affinity chromatography with an Ni2ϩ-nitrilotriacetic acid column in 0.1 M Tris/ HCl, 20 mM imidazole, pH 7.4, binding buffer with stepwise elution up to 500 mM imidazole. 1 M iodoacetamide in 1 M Tris/HCl, pH 8.5, 20 mM EDTA was added at a 1:5 (v/v) ratio to the protein solution, and the reaction was performed for 15 min at 20 °C. Measurements were performed in 0.1- and 0.5-mm quartz cells with 0.5 g/liter protein samples in 20 –50 mM sodium phosphate, 20 mM NaCl, pH 7.2, at 4 °C. The atomic coordinates have been deposited in the Protein Data Bank [34], accession code 3E07
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