ABSTRACT
Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases. The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography. Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined. The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution. This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM). The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme. Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin. Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain. This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors. CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM). This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Oligopeptides/chemistry , Oligopeptides/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Renin/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Hydrogen Bonding , Kinetics , Models, Molecular , Oligopeptides/pharmacology , Protease Inhibitors/pharmacology , Protein Structure, Secondary , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins , Static Electricity , Substrate Specificity , ThermodynamicsABSTRACT
Big ET-1 and big ET-3 are precursor peptides which render endothelin-1 (ET-1) and endothelin-3 (ET-3) relatively unreactive and resistant to proteolytic cleavage. Big ET-1 is cleaved in vivo by ECE-1 (endothelin-converting enzyme), and big ET-3 is also cleaved but apparently to a significantly lesser extent by this enzyme. To shed light on the relation between structure and function, circular dichroism (CD) spectroscopy and homology modeling were used to determine whether big ET-1 and big ET-3 adopt similar secondary and tertiary structures. Analyses of the CD spectra and thermal denaturation indicate they have similar secondary structures and thermal stabilities. Superposition of the modeled coordinates of both peptides indicates that they can adopt the same overall fold except in the C-terminal residues, 34-38 in big ET-1 and 34-41 in big ET-3. This region corresponds to an area of complete sequence heterogeneity between the two peptides. A model has been developed which has a loop for residues 27-30 (HVVP in big ET-1), which have previously been demonstrated to be essential for eliciting efficient hydrolysis of the W21-V22 bond in big ET-1 and which have the sequence QTVP in big ET-3. Differences in affinity between big ET-1 and big ET-3 for ECE-1 thus appear to be due solely to sequence variations in the local region of the cleavage site.
Subject(s)
Aspartic Acid Endopeptidases/chemistry , Endothelins/chemistry , Protein Folding , Protein Precursors/chemistry , Amino Acid Sequence , Animals , Cattle , Circular Dichroism , Endothelin-1 , Endothelin-3 , Endothelin-Converting Enzymes , Guinea Pigs , Humans , Hydrogen-Ion Concentration , Metalloendopeptidases , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Rabbits , Rats , Sequence Homology, Amino Acid , Substrate Specificity , TemperatureABSTRACT
The crystal structures of glycosylated native proteinase A, an aspartic proteinase found in the vacuole of Saccharomyces cerevisiae, and its complex with a difluorostatone-containing tripeptide have been determined by molecular replacement to 3.5 A and 2.4 A resolutions, respectively. Superposition of the bound and native forms gave an r.m.s. difference of 0.6 A largely reflecting the poor resolution of the native crystal structure. The secondary and tertiary structures are highly similar to those found in porcine pepsin and lysosomal cathepsin D; superposition of the structure of proteinase A bound to the difluorostatone inhibitor on those of pepsin and cathepsin D gave pairwise r.m.s. differences for C(alpha) atoms of 1.36 A and 0.88 A. Most differences occur in loop regions. Comparison of the structure of the proteinase A-difluorostatone complex with that of endothiapepsin bound with the same inhibitor shows that the conformation and hydrogen bond interactions of the inhibitor in the active site are very similar, even though the enzymes have only 27% sequence identity. Electron density for the crystal structure of the proteinase A complex reveals five residues of the oligosaccharide structure attached to Asn67: Man-(1 --> 2)-alpha-Man-(1 --> 3)-beta-Man-(1 --> 4)-beta-GlcNAc-(1 --> 4)-beta-GlcNAc-Asn-67. The first three residues of the oligosaccharide cover the same region of the protein surface as those of the oligosaccharide attached to the equivalent position in cathepsin D. The second carbohydrate attachment site is disordered beyond the first carbohydrate residue in both enzymes.
