Abstract
Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys(13)) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.
Highlights
Kunitz-type inhibitors provide a suitable scaffold for novel elastase inhibitors
Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type inhibitors toward elastase-like enzymes
A comparison with other canonical inhibitors in complex with human neuthrophil elastase (HNE) and porcine pancreatic elastase (PPE) revealed that the side chain of Arg[11] at the P3 site of ShPI-1/K13L extends into the same enzyme region as the P5 residue of chelonians, which is reported to direct the elastase selectivity of these inhibitors (12)
Summary
Kunitz-type inhibitors provide a suitable scaffold for novel elastase inhibitors. Results: The inhibitor ShPI-1 was modified for pancreatic elastase binding, and the crystal structure of the complex was elucidated and analyzed. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes. BPTI-Kunitz inhibitors usually contain a basic residue at the reactive site, denoted as P1 position by Schechter and Berger (20) They strongly inhibit trypsin-like enzymes, and chymotrypsin and HNE, with comparatively lower affinity. A comparison with other canonical inhibitors in complex with HNE and PPE revealed that the side chain of Arg[11] at the P3 site of ShPI-1/K13L extends into the same enzyme region as the P5 residue of chelonians, which is reported to direct the elastase selectivity of these inhibitors (12). Supporting a previous report of a BPTI variant, our structural results will significantly contribute to selectivity modifications of BPTI-Kunitz-type inhibitors toward these enzymes in the future
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
