Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster

Abstract

The Spätzle protein is involved in both the definition of the dorsal-ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 A resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P2(1)2(1)2(1), with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 A) that diffracted to 2.8 A resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.

Figure 1

Crystals of Spz11.7 from 50 mM Tris–HCl, 20% PEG 3350 pH 8.0 with 10 mM betaine monohydrate as an additive.

Figure 2

Silver-stained SDS–PAGE of Spz11.7 used for crystallization (lane S, starting material), a crystallization drop (lane D) and a dissolved crystal (lane C); Lane M, molecular-weight markers (kDa).

Figure 3

Nonreducing SDS–PAGE of Spz11.7 crystallization screening experiments using 4% PEG 3350 as precipitant and 0.05 M buffer. The pH values of individual crystallization drops are indicated and were achieved using the following buffers: sodium citrate (E1–F5), MES (F9), bis-tris–HCl (G1), imidazole–HCl (G5), sodium HEPES (G9) and Tris–HCl (H1–H5). Note the varying degradation bands observed. Lanes E9, F9 and H1 exhibit comparable bands (∼25 kDa) to those present in the crystals (see Fig. 2 ▶). Marked gel slices from E9, F9 and H1 were further analysed using MALDI-MS (see Fig. 4 ▶).

Figure 4

Recombinant Spz11.7 sequence, indicating the N-terminal propeptide as well as the mature cystine knot (bold). The peptides detected by MALDI-MS are underlined in black (from sample H1), red (sample F9), gold (sample E9, high-molecular-weight band) or grey (negative control, E9 low-molecular-weight band, see text for details). The dashed line indicates a peptide with ambiguous assignment (peptide mass ⁵⁵Y–R⁶⁶, 1437.807 Da; ²⁰⁵S–K²¹⁷, 1437.686 Da).