Crystal Structure of an Elastase-Specific Inhibitor Elafin Complexed with Porcine Pancreatic Elastase Determined at 1.9 Å Resolution

Abstract

The crystal structure of a stoichiometric complex between an elastase-specific inhibitor elafin and porcine pancreatic elastase (PPE) has been determined and refined to a crystallographic R-factor of 19.7% at 1.9 A resolution. The polypeptide chain of elafin has a planar spiral shape with an exposed external part and an internal core part which resembles both the crystal structure of human seminal plasma inhibitor (HUSI-1) [Grütter, M. G., Fendrich, G., Huber, R., & Bode, W. (1988) EMBO J. 7, 345-351] and the solution structure of Na+,K(+)-ATPase inhibitor (SPAI-1) revealed by NMR analysis [Kozaki, T., Kawakami, Y., Tachibana, S., Hatanaka, H., & Inagaki, F. (1994) Pept. Chem., 405-408]. The external region containing the primary binding loop is interconnected by four disulfide bonds to the internal part composed of a beta-sheet and a hairpin loop. The scissile peptide bond Ala24i(P1)-Met25i(P1') in the primary binding site is intact, and its carbonyl carbon is in van der Waals contact with O gamma of the active site Ser195 of PPE. The seven residues of Leu20i(P5)-Leu26i(P2') of the primary binding loop and the three residues of Ser48i, Cys49i, and Ala52i of the adjacent hairpin loop are in contact with PPE by hydrogen bonds and/or van der Waals interactions in a manner similar to that observed for other serine protease-inhibitor complexes. Electron densities of the N-terminal residues Ala1i-Ser10i which are not responsible for the elastase inhibitory activity were not visible, probably due to disordered conformation. The guanido group (N eta 1, N eta 2) of Arg61 in the complex interacts with S delta of Met25i(P1') by possible hydrogen bonds between N and S atoms, accompanying a large positional shift of the side chain of Arg61-(S1') between the complexed and free forms of PPE. The primary binding site is stabilized by hydrogen bonds between the guanido group (N eta 1, N eta 2) of Arg22i(P3) and the carbonyl group of Met25i(P1') across the scissile bond, as well as by a hydrogen bond between the amino group of Cys23i(P2) and the carbonyl group of Ser48i in the internal core. This intramolecular hydrogen bond network and the network of four disulfide bonds might play a significant role in stabilizing the conformation of the binding site for expressing the potent specific inhibitory activity.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

“…We also found a partial cleavage in the disulfide core (at Ala-24 -Met-25) mediated by NE within 24 h of incubation using elafin in slight excess (data not shown). The x-ray crystallographic analysis of elafin complexed with porcine pancreatic elastase (37) indicated that this peptide bond is located in the inhibitory loop of elafin and corresponds to the scissile peptide bond, also called P1-P1Ј bond, according to the nomenclature of Schechter and Berger (45). Such a cleavage occurring at the scissile bond can be observed for protease inhibitors that obey the standard mechanism described by Laskowski et al (46).…”

“…The ability of excessive NE to cleave elafin suggests that NE preferentially interacts with the tprotease-binding loop rather than cleaving the N-terminal extremity of elafin. Structural studies of elafin (37,38) indicated that the N-terminal extremity of the inhibitor is at the opposite side of the protease binding loop (inhibitory loop). Therefore, free NE can likely cleave the inhibitor complexed with its target enzyme.…”

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

“…However, leukocyte elastase itself may not be the endogenous target of guinea pig trappin because the affinity of the recombinant WAP motif for leukocyte elastase (K i ϭ 2.33 ϫ 10 Ϫ7 M) is much lower compared with those of other WAP motif proteins such as recombinant elafin (K i ϭ 1.27 ϫ 10 Ϫ8 M), SLPI (K i ϭ 6.3 ϫ 10 Ϫ11 M), and trappin-2 (elafin) (K i ϭ 1.7 ϫ 10 Ϫ10 M) (1). In support of this speculation, the amino acid sequences of the variable region of the WAP motif are quite different between guinea pig trappin and the elastase inhibitors SLPI and trappin-2 (elafin) (1); the variable region has been shown to be the site of interaction with the target proteinase by x-ray crystallographic analysis of an elastase⅐trappin-2 (elafin) complex (45). Oxidation of Met 14 and Met 41 in human elafin (Fig.…”

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

“…A critical structure/function role for loop regions has also been suggested for other high-affinity protease inhibitors, most notably those of the chelonianin family that includes the endogenous, noncovalent NE inhibitors elafin (23) and SLPI (24). Besides the insertion of an inhibitory loop region into the NE active site, however, the SLPI/NE complex has little in common with the EapH1/NE complex (Fig.…”