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Human neutrophil elastase (hNE) is an abundant serine protease that is a major constituent of lung elastolytic activity. However, when secreted in excess, if not properly attenuated by selective inhibitor proteins, it can have detrimental effects on host tissues, leading to chronic lung inflammation and non-small cell lung cancer. To improve upon the design of inhibitors against hNE for therapeutic applications, here, we report the crystal structure of hNE in complex with an ecotin (ET)-derived peptide inhibitor. We show that the peptide binds in the nonprime substrate binding site. Unexpectedly, compared with full-length (FL) ET, we find that our short linear peptides and circular amide backbone-linked peptides of ET are incapable of efficient hNE inhibition. Our structural insights point to a preferred amino acid sequence and the potential benefit of a scaffold for optimal binding and function of the peptide inhibitor, both of which are retained in the FL ET protein. These findings will aid in the development of effective peptide-based inhibitors against hNE for targeted therapy.

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.