Solution conformation of a synthetic bis-headed inhibitor of trypsin and carboxypeptidase A: new structural alignment between the squash inhibitors and the potato carboxypeptidase inhibitor.
The trypsin carboxypeptidase peptide inhibitor (TCPI) which inhibits both trypsin and carboxypeptidase A has been chemically engineered by modification of the Ecballium elaterium trypsin inhibitor II (EETI-II). The solution conformation of TCPI, studied by two-dimensional nuclear magnetic resonance, was shown to be very close to those of squash inhibitors. Only limited deviations of the trypsin binding loop compared to its location in the EETI-II/trypsin complex were detected. It was also shown that the position of the C-terminal tail did not significantly change from the position observed in the complex between carboxypeptidase A and the potato carboxypeptidase inhibitor (PCI). The conformation of TCPI was carefully compared with the PCI one and a new structural alignment between the two microproteins is proposed. This alignment points out the very good conservation in the two inhibitors of a subdomain comprising segments 7-15, 19-22 and 25-28. Most importantly, the 2-19 disulfide bridge of TCPI was not structurally conserved in PCI and appeared to be rather unimportant for the early folding process of these molecules. This result agrees with the recent observation that the 2-19 bridge is the last to be formed in the folding of the squash inhibitor EETI-II and suggests that this is also the case during the folding of the potato carboxypeptidase inhibitor.
Chiche
L
L
Centre de Biochimie Structurale, Faculté de Pharmacie, Montpellier, France.
Heitz
A
A
Padilla
A
A
Le-Nguyen
D
D
Castro
B
B
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010944:Plants / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D014361:Trypsin Inhibitors / Q000737:chemistry*; D014675:Vegetables
D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012996:Solutions; D014361:Trypsin Inhibitors; C084638:trypsin carboxypeptidase peptide inhibitor; D002268:Carboxypeptidases; D043422:Carboxypeptidases A