3e 28, SSG two has the motifs encoding the GTPase domain using the corresponding consen sus sequences concerned in GTP binding shaded in gray in Figure 1B. The phosphate binding loop which includes the sequence GXGXXGKS is noticed in SSG two as GSGES GKS. The magnesium binding residues using the consen sus sequence DXXG is current as DVGG in SSG two, although the guanine ring binding web sites are individuals together with the consen sus sequence NKXD is present as NKVD. The TXAT con sensus sequence is current as TQAT in SSG 2. Another area concerned in phosphate binding incorporates the con sensus sequence RXXT that in SSG two is present as RTKT. Along with these conserved domains, the protein derived from the ssg 2 cDNA sequence has the N terminal glycine that is definitely myristoylated in G subtypes and is essential for membrane association. The 5 residues that recognize the adenylate cyclase interaction web-site according to BLAST anal ysis are in red in Figure one, these include I187, K212, I215, H216, and E 219.
The putative receptor binding webpage involves amino acids L318 to R334 and is shown in blue letters in Figure one. The derived amino acid sequence alignment of SSG 2 to that of your a few fungal homologues is shown in Figure 2. This figure demonstrates even more than 85% identity to MAGA of M. grisea, CPG two of C. parasitica PFT �� and GNA three of N. crassa, Table 1 summarizes the percent identity of SSG 2 to some members with the fungal G homologues and SSG one. Yeast two hybrid screening Two independent yeast two hybrid screenings, applying dif ferent S. schenckii yeast cells cDNA libraries have been executed using the full coding sequence of SSG two as bait. In both screenings, 3 blue colonies developing in quadruple drop out selleckchem medium have been recognized as containing the exact same PLA2 homo logue insert.
The expression with the Ade, His phenotypes and galactosidase activity are regarded by the manu facturer as corroborative of true interactions. The inserts from all 3 colonies have been found to incorporate the carboxy terminal residues of the protein homologous to PLA2s from A. nidulans. Our success indicated the final 162 amino acids within the S. schenckii cPLA2 homologue interacted with SSG 2. The SSG 2 SSPLA2 interaction was corroborated by co immunoprecipitation. Figure three shows the confirmation from the interaction observed from the yeast two hybrid assay in between SSG two and SSPLA2 by co immunoprecipitation and Western blot analysis. Lane one shows the band obtained working with anti cMyc antibody that recognizes SSG two. This band is with the expected dimension contemplating that SSG two was expressed fused to the GAL 4 binding domain. The two higher molecular bodyweight bands existing belong to the anti cMyc antibodies used for precipitation. Lane 2 demonstrates the outcomes obtained within the Western blot when the amino acid sequence are, respectively.