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

Abstract

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.

Keywords: elastase; inhibition; peptide; serine protease; structure.

FIGURE 1

Design of peptides. Peptides were derived from ecotin, a potent inhibitor of human neutrophil elastase (hNE). Four peptides were designed for the inhibition studies. (a) Pep1 is a linear peptide with the sequence ⁷⁷VSSPVSTM⁸⁴. Pep2 is an amide‐cyclized peptide with the same sequence as Pep1. The N and C terminals are covalently linked through an amide bond. The Pep2 was designed to ensure lower protease digestion, expose the residues, and facilitate interaction with hNE. The Pep3 is a longer linear peptide with the sequence ⁷⁷VSSPVSTMMACPA⁸⁹. This peptide was designed to occupy the prime subsites beyond S1, taking cues from the previously known structure (7CBK, hNE‐ecotin complex). Similar to Pep2, Pep4 is a cyclized peptide of Pep3 designed to maintain the stability of the peptide for better inhibition. (b) The four peptides (Pep1–Pep4) were used in enzymatic assays to evaluate their inhibitory potencies against wild‐type hNE. The chromogenic substrate, upon cleavage by hNE, releases p‐nitroaniline that is detected at 405–410 nm. The inhibition curves shown here indicate that the full‐length ecotin protein has the best inhibition at 0.7 nM. Out of the four peptides, Pep1 has the best IC₅₀ of 1.9 μM followed by Pep3 with 10.8 μM. Pep2 and Pep4 were designed as amide‐cyclized peptides for better stability and interaction with hNE. However, they seem to have very low inhibitory effect on the activity of the enzyme

FIGURE 2

Crystal structure of hNE‐ET^Pep1. (a) The left panel shows the hNE‐ET^Pep1 monomer structure and the Pep1 bound to the S subsites of hNE enzyme. The 2Fo − Fc map (gray isomesh) for the bound peptide (magenta) is contoured at 0.9 σ. The hNE (shown in as green cartoon) is glycosylated at two amino acids (shown in cyan)—Asn109 and Asn159. The right panel shows the bound peptide consisting of Met84, Thr83, Ser82, and Val81 which are P1–P4 positions of the inhibitor, and they interact with the S1–S4 subsites of the hNE (shown as green surface representation). The catalytic triad (His57, Asp102, and Ser195) is shown in cyan. (b) Comparison of the peptide orientation from known hNE complex crystal structures. There are four known structures of hNE‐peptide complexes (1HNE, 1PPG, 2RG3, and 4WVP). These peptides in these structures target the same subsites on hNE as Pep1 from our structure. hNE is shown as an electrostatic potential surface with the peptides aligned well. The P1 and P2 target the S1 and S2 subsites which are largely acidic in nature, whereas the P3 and P4 target S3 and S4 subsites which form more basic/alkaline regions in hNE. The superposition of the hNE‐peptide complexes shows that the peptides occupy the same subsites in the hNE (electrostatic surface representation). The catalytic triad residues (shown in dark gray) are close to the S2 subsites. (c) The Pep1 (shown as thick sticks in magenta) interacts with S1–S4 subsites formed by the residues of hNE (thin green sticks). The key hydrogen bonds are shown as thin black dashed line

FIGURE 3

The scaffold of the inhibitor is crucial. (a) The structural superposition of hNE‐ET^Pep1, hNE‐ET (human neutrophil elastase–ecotin) and the known hNE‐peptide complexes (PDB codes 1HNE, 1PPG, 2RG3, 4WVP) show that the peptides bind to an active site groove on hNE. The groove is shown in different orientations. Left side view, central view and right‐side view are shown here. Primarily, the groove consist of all residues (⁴¹CG⁴², ⁵⁶HC⁵⁷, ⁹⁵NL⁹⁶, ¹⁶¹LCR¹⁶³, ¹⁷⁶VCFGDSGS¹⁸³, ¹⁹⁵ASFVRGG²⁰¹ and ²⁰⁶Y) of hNE within 5 Å distance from the inhibitory peptides (shown as sticks in the central view right panel). The ecotin inhibitory region (shown in grey) traverses this hNE groove deeply and therefore responsible for the effective nanomolar inhibition of the enzyme. In the bottom right panel, the surface is shown to be transparent with the Pep1 (magenta sticks) and ecotin (grey ribbon). (b) The comparison of the hNE‐ET^Pep1, hNE‐ET and the known peptide complexes (PBD codes 1HNE, 1PPG, 2RG3, 4WVP) are shown here. The amino acids at P4 to P3′ positions from different peptides (and FL ecotin) are shown. The superposition and related RMSD (root‐mean‐square deviation) values show that the structures align well. The buried surface area (BSA) is the highest in the ecotin‐hNE complex followed by hNE‐ET^Pep1 and then other peptides complexes. The number of hydrogen bonds between the inhibitor peptide (or ecotin protein) and hNE protein is shown in the last row. The figures below correspond to the inhibitor present in the respective structure