Prediction of the biologically active sites in eclosion hormone from the silkworm, Bombyx mori

Abstract

The structure-activity relationship of eclosion hormone from the silkworm, Bombyx mori, was analyzed. First, the probable active residues in silkworm eclosion hormone and also tobacco hornworm eclosion hormone were predicted by the average distance map method. To examine the contributions of those residues to the activity of silkworm eclosion hormone, Gly-substituted mutants for those predicted residues were produced by site-directed mutagenesis and their activities were evaluated by a bioassay. Finally, Glu12, Met24 and Phe25 were estimated to be the crucial residues for the eclosion hormone activity. The possibility of the development of a blocker of an eclosion hormone receptor on the basis of the present work is also discussed.

De Novo Molecular Modeling and Biophysical Characterization of Manduca sexta Eclosion Hormone

“…Kono et al (14) reported that removal of the first six N-terminal residues (an unordered region in our model) had little effect on activity, while cleavage of residues from the hydrophobic C-terminus dramatically reduced activity. Similar results were seen with Gly substitution of Met24, Phe25, Phe29, Lys40, Pro47, Glu50, Ile55, Phe58, and Leu59 (18, 19). The conservatively substituted hydrophobic residue at position 25 (Phe or Tyr in over 60% of identified sequences) was postulated to have hydrophobic interactions with Phe58 (18), but in our model the side chains of these two residues fail to meet the distance criteria for such an interaction (Figure 11).…”

“…Structure-Function Analyses of Point Mutations. Previous studies (18,19) reported the effects of 29 Gly point mutations on the potency of Bombyx EH, which demonstrated the particular importance of Met24, Phe25, Phe29, Ile55, Phe58, and Leu59. On the basis of those studies, Phe25 (Leu in Manduca), Phe58, and Leu59 were thought to play important roles in stabilizing the EH globular structure, Phe29 and Ile55 were thought to be necessary for receptor interactions, and Met24 was thought to play a role in both functions.…”

“…An intact C-terminus, in contrast to the N-terminus, is also important for activity , . Point mutations in Bombyx EH have identified a number of residues that are important for biological activity, including conserved residues Met24, Phe29, Ile55, Pro47, Phe58, and Leu59, as well as a hydrophobic residue at position 25 and a hydrophilic residue at position 40 , . Despite these structure−function analyses, little progress has been made in elucidating the EH solution structure.…”

Analysis of the differences in the folding kinetics of structurally homologous proteins based on predictions of the gross features of residue contacts

“…Originally, this method was developed for the prediction of location of domains. However, it can also be applied to the various problems related to the 3D properties of proteins (e.g., similarity of 3D structures, packing arrangements in β‐sheets,30 local compact regions,31 active sites, or receptor recognition sites in bioactive peptides) 32–34. A constructed map based on this method predicts the location of folding nuclei in the sequence of a protein and might contain information about the early events of the folding process of a protein.…”