The covalent structure of the elastase inhibitor from Anemonia sulcata--a "non-classical" Kazal-type protein.
The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003063:Cnidaria / Q000378:metabolism*; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis; D006868:Hydrolysis; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000032:analysis; D012615:Sea Anemones / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000032:analysis*; D014361:Trypsin Inhibitors / Q000032:analysis*
D000596:Amino Acids; D004220:Disulfides; D010455:Peptides; D014361:Trypsin Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic; D010196:Pancreatic Elastase; D003488:Cyanogen Bromide