The crystal structure of JNK from Drosophila melanogaster reveals an evolutionarily conserved topology with that of mammalian JNK proteins.

BACKGROUND: The c-Jun N-terminal kinases (JNKs), members of the mitogen-activated protein kinase (MAPK) family, engage in diverse cellular responses to signals produced under normal development and stress conditions. In Drosophila, only one JNK member is present, whereas ten isoforms from three JNK genes (JNK1, 2, and 3) are present in mammalian cells. To date, several mammalian JNK structures have been determined, however, there has been no report of any insect JNK structure. RESULTS: We report the first structure of JNK from Drosophila melanogaster (DJNK). The crystal structure of the unphosphorylated form of DJNK complexed with adenylyl imidodiphosphate (AMP-PNP) has been solved at 1.79 Å resolution. The fold and topology of DJNK are similar to those of mammalian JNK isoforms, demonstrating their evolutionarily conserved structures and functions. Structural comparisons of DJNK and the closely related mammalian JNKs also allow identification of putative catalytic residues, substrate-binding sites and conformational alterations upon docking interaction with Drosophila scaffold proteins. CONCLUSIONS: The DJNK structure reveals common features with those of the mammalian JNK isoforms, thereby allowing the mapping of putative catalytic and substrate binding sites. Additionally, structural changes upon peptide binding could be predicted based on the comparison with the closely-related JNK3 structure in complex with pepJIP1. This is the first structure of insect JNK reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of insect JNK.

Crystal structure of BinB: a receptor binding component of the binary toxin from Lysinibacillus sphaericus.

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N- and C-termini. The globular N-terminal domain has a ß-trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor-binding. The BinB ß-rich C-terminal domain shares similar three-dimensional folding with aerolysin type ß-pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin-like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation.

Crystallization and preliminary X-ray crystallographic analysis of the functional form of BinB binary toxin from Bacillus sphaericus.

The binary toxin from Bacillus sphaericus consists of two proteins, BinA and BinB, which work together to exert toxicity against mosquito larvae. BinB is proposed to be a receptor-binding domain and internalizes BinA into the midgut cells, resulting in toxicity via an unknown mechanism. The functional form of BinB has been successfully crystallized. The crystals of BinB diffracted to a resolution of 1.75 Å and belong to space group P6(2)22, with unit-cell parameters a = b = 95.2, c = 154.9 Å. Selenomethionine-substituted BinB (SeMetBinB) was prepared and crystallized for experimental phasing. The SeMetBinB crystal data were collected at a wavelength of 0.979 Å and diffracted to a resolution of 1.85 Å.

Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis.

The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)(6)-tagged BinA and (His)(6)-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni-NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC(50) of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly ß-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation.