Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from
Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized
![](/images/gifchars/alpha.gif)
-helical(CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solutionstructure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by acentral
![](/images/gifchars/alpha.gif)
-helix and a three-stranded antiparallel
![](/images/gifchars/beta2.gif)
-sheet of a typical Kazal-type inhibitor such as silverpheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forminga disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3.Synthesized AEI exhibited unexpected strong inhibition toward
Streptomyces griseus protease B (SGPB).Our previous study [
Hemmi, H., et al. (2003)
Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motifcould produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI(AEI analogue) was chemically synthesized when a Cys
4-Cys
34 bond was changed to a Cys
6-Cys
31bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibitedalmost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structuraldifference was detected between the two, but the N-terminal loop from Pro
5 to Ile
7 near the putativereactive site (Met
10-Gln
11) in the analogue moved by 3.7 Å toward the central helix to form the introducedCys
6-Cys
31 bond. Such a conformational change in the restricted region correlates with the specificitychange of the inhibitor.