Structural Basis for the Inhibition Mechanism of Ecotin against Neutrophil Elastase by Targeting the Active Site and Secondary Binding Site

Abstract

Human neutrophil elastase (hNE) is a serine protease that plays a major role in defending the bacterial infection. However, elevated expression of hNE is reported in lung and breast cancer, among others. Moreover, hNE is a target for the treatment of cardiopulmonary diseases. Ecotin (ET) is a serine protease inhibitor present in many Gram-negative bacteria, and it plays a physiological role in inhibiting host proteases, including hNE. Despite this known interaction, the structure of the hNE−ET complex has not been reported, and the mechanism of ecotin inhibition is not available. We determined the structure of the hNE−ET complex by molecular replacement method. The structure of the hNE−ET complex revealed the presence of six interface regions comprising 50s, 60s, and 80s loops, between the ET dimer and two independent hNE monomers, which explains the high affinity of ecotin for hNE (12 pM). Notably, we observed a secondary binding site of hNE located 24 Å from the primary binding site. Comparison of the closely related trypsin−ecotin complex with our hNE−ET complex shows movement of the backbone atoms of the 80s and 50s loops by 4.6 Å, suggesting the flexibility of these loops in inhibiting a range of proteases. Through a detailed structural analysis, we demonstrate the flexibility of the hNE subsites to dock various side chains concomitant with inhibition, indicating the broad specificity of hNE against various inhibitors. These findings will aid in the design of chimeric inhibitors that target both sites of hNE and in the development of therapeutics for controlling hNE-mediated pathogenesis.

Sequence preference and scaffolding requirement for the inhibition of human neutrophil elastase by ecotin peptide

“…This accessory interaction may facilitate a more efficient binding and, in turn, inhibition of the protein. 17,30 In our investigations-and contrary to many previous reports 20,21,23 -the cyclized peptides had no enhanced hNE inhibition over the linear peptides, indicating that the amide cyclization of these peptides did not favorably orient the P4-P1 (or P4-P4 0 in the case of the larger peptide, Pep3) amino acids to enhance hNE inhibition. We surmise that a larger cyclic peptide might offer an improved binding orientation.…”

“…ET is a potent inhibitor of hNE. 19 Using our previous complex structure and guided by literature, 17,20,21 we designed four ET-derived peptides (Pep1-Pep4) and tested their inhibitory potential against hNE. Peptide 1 (Pep1) was designed as a linear peptide to interact with the non-prime S site, whereas the linear peptide 3 (Pep3) was designed to interact with both the non-prime and prime, S and S 0 sites, respectively (See Materials and Methods, Section 3).…”

“…In the current complex structure, Pep1 occupies the same subsites as ET in the hNE-ET complex previously solved by us (7CBK). 17 Notably, the peptide consists of Val81 (P4), Ser82 (P3), Thr83 (P2), and Met84 (P1) interacting with the S4 (Val198), S3 (Val198), S2 (Ser196, Ser181, and His56), and S1 (Ser181, Gly179, Phe178, Cys177, and His57) subsites of hNE, respectively. Pep1 forms several hydrogen bonds with the hNE protein, the complex further strengthened by numerous hydrophobic interactions.…”

“…These peptides were designed based on our previous study and literature. 17,18 We also report the crystal structure of Pep1 in complex with hNE. Compared with ET, we observe that these inhibitors cannot fully inhibit hNE.…”

“…A recent report from Hochscherf et al detailed the structure of a small-molecule inhibitor (1,3-thiazolidine-2,4-dione derivative) in complex with leukocyte elastase (PDB code 6F5M), in which the inhibitor targeted the prime site of the substrate binding site. 16 Recently, we reported the crystal structure of hNE in complex with the serine protease inhibitor protein, ecotin (ET; MW 18.3 kDa) 17 that lead to the identification of two independent binding regions of ET involved in the inhibition of hNE. Further, the comparative analyses revealed how the hNE activity can be inhibited by various inhibitors and the broad specific inhibition property of ET against various proteases.…”