Molecular recognition between serine proteases and new bioactive microproteins with a knotted structure.
Microproteins with proteinase inhibitory activity, 28 to 30 amino acids long, with 3 disulfide bridges have been isolated from Ecballium elaterium seeds. A peptide (EETI II) was isolated and behaved as a powerful trypsin inhibitor (Kd = 10(-11) to 10(-12) M). It was sequenced, chemically synthesized and the 3-D structure determined by 2-D 1H NMR. The information gained in the process enabled us to synthesize modified derivatives with inhibitory activity towards pancreatic elastase, chymotrypsin and human leucocyte elastase (Kd = 10(-7) to 10(-9) respectively). The most striking characteristic that appeared during the synthetic approach was the unfailing ability of the 28 amino acid peptides to refold and correctly close the 3 disulfide bridges, giving in each case an active compound. These disulfide bridges are assembled in a particular knotted structure, shared by few other bioactive peptides and called the 'knottin' structure. Molecular modeling of the peptide and a comparison with the other active molecules with similar topology allowed the synthesis of a chimaeric peptide, bearing 1 active site against a seryl-protease (trypsin), and 1 against a metallo-protease (carboxypeptidase A). The bis-headed peptide was able to inhibit both enzymes separately and concomitantly.
Le Nguyen
D
D
Centre CNRS-INSERM de Pharmaco-Endocrinologie, Montpellier, France.
Heitz
A
A
Chiche
L
L
Castro
B
B
Boigegrain
R A
RA
Favel
A
A
Coletti-Previero
M A
MA
D000595:Amino Acid Sequence; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D010940:Plant Proteins; D014361:Trypsin Inhibitors; C063950:trypsin inhibitor EETI II protein, plant