Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L

Abstract

The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, K i = 2.35·10−8 M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (K i = 1.3·10−9 M, and K i = 1.2·10−8 M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor’s P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases.

The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels

“…The mode of accommodation in the catalytic pocket of HNE is still unknown, but molecular modeling studies have suggested that polar desolvation and the presence of Asp226 in HNE could favor the interaction with the basic P1 residue. These interactions cannot be established by Thr226 of PPE, which precludes the insertion of K13 side-chain into the S1 subsite of this enzyme [ 29 ], thus explaining the absence of activity against this enzyme.…”

Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa

“…Compared to porcine pancreatic elastase (PPE), the S1 pocket of HNE is more flexible and is negatively charged5253, which may explain the recognition of Lys or Arg at the P1 position of the inhibitor, leading to the selectivity of biotin-rT1 towards HNE (supplementary data S10). The S2 pocket of HNE is hydrophobic and can accept peptidyl inhibitors with Pro residue at its P2 position545556.…”

Molecular Characterization of Kunitz-Type Protease Inhibitors from Blister Beetles (Coleoptera, Meloidae)

“…The second group has hydrophobic side chains at the P1 site. The presence of a Leu residue at the P1 site gives a higher affinity for elastases depending on stronger pairwise van der Waals interactions and less unfavourable polar-desolvation drawback compared to inhibitors with Lys residues at P1 [ 58 ]. Moreover, the carbonyl oxygen atom of Leu13 occupies the oxyanion hole, establishing three hydrogen bonds with the backbone nitrogen atoms of Elastase residues Gly193, Asp194, and Ser195, together with an H-bond with Ser214 via its nitrogen atom.…”