Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction

Abstract

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity.

In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates

“…ITPs occur in insects, whereas CHH, MIH, MOIH, and GIH occur only in decapods ( 8 , 22 , 77 – 79 ). These large neuropeptides have a unique compact core structure consisting of four or five α-helical regions and stabilized by three intramolecular disulfide bridges ( 3 , 9 , 12 , 13 ). However, differences in N- and C-terminal sequences, chemical modifications, and distribution of surface amino acid residues confer ligand/receptor binding affinity and specificity.…”

“…No isoforms are generated by alternative splicing in type II peptides and post-translational modifications are uncommon ( 3 , 5 ). CHH superfamily mature peptides have a compact native conformation stabilized by the three disulfide bridges and nine conserved hydrophobic residues ( 3 , 9 , 12 , 13 ). Type I peptides have four α-helices and type II peptides have the four α-helices and an additional short α1/3 10 -helix located around the conserved Gly12 ( 5 , 9 ).…”

“…Type I peptides have four α-helices and type II peptides have the four α-helices and an additional short α1/3 10 -helix located around the conserved Gly12 ( 5 , 9 ). Functional studies of expressed mutant MIH recombinant constructs show that both the N- and C-terminal regions, which come in close apposition in the native structures, contribute to MIH activity ( 9 , 13 , 14 ). Interestingly, the two residues at positions #13 and #14 in the α1 helix, but not the Gly12 itself, are critical for full MIH activity ( 9 , 14 ).…”