Abstract
Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.
Highlights
Chymotrypsin C (CTRC) targets specific regulatory cleavage sites within trypsinogens and procarboxypeptidases
Structure Solution and Refinement—The structure of active human chymotrypsin C (CTRC) bound to inhibitor eglin c was determined to 1.9-Å resolution in space group P212121 with one bimolecular complex per asymmetric unit
The structure was solved by molecular replacement using as search models the previously reported structures of porcine pancreatic elastase (PDB 1EAI) [21], featuring 52% sequence identity to human CTRC, and eglin c (PDB 1ACB) [22]
Summary
Chymotrypsin C (CTRC) targets specific regulatory cleavage sites within trypsinogens and procarboxypeptidases. Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Several mutations in the CTRC gene itself that lead to loss or impairment of protein function are significantly associated with chronic pancreatitis (9 –12) Both cationic trypsin and CTRC are members of the chymotrypsin family of serine peptidases, which share a common two -barrel-fold, a famous triad of catalytic residues Ser, His, and Asp, and a conserved catalytic mechanism for nucleophilic cleavage of peptide bonds. Analysis of the structure suggests that whereas the bulk of binding energy derives from burial of the P1 residue and several other hydrophobic side chains, specificity may derive largely from the exaggerated role of long-range electrostatic interactions, from a moderate preference for Leu at the P1 position, and may be influenced by local sequence-dependent backbone conformational tendencies
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