Molecular recognition between serine proteases and new bioactive microproteins with a knotted structure

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ecballium elaterium seeds contain microproteins with proteinase inhibitory activity.
• These peptides are 28-30 amino acids long and feature 3 disulfide bridges.

Purpose of the Study:

• To isolate, characterize, and synthesize novel proteinase inhibitors.
• To investigate the structure-activity relationship of these peptides.
• To develop modified peptides with broader inhibitory profiles.

Main Methods:

• Isolation and sequencing of peptides from Ecballium elaterium seeds.
• Chemical synthesis and 3-D structure determination using 2D 1H NMR.
• Molecular modeling and synthesis of modified and chimeric peptides.

Main Results:

• A potent trypsin inhibitor (EETI II) was identified with Kd = 10(-11) to 10(-12) M.
• Modified derivatives showed inhibitory activity against pancreatic elastase, chymotrypsin, and human leukocyte elastase (Kd = 10(-7) to 10(-9) M).
• Synthesized peptides refolded correctly, forming active compounds with a conserved 'knottin' structure.
• A chimeric peptide inhibited both trypsin and carboxypeptidase A.

Conclusions:

• Ecballium elaterium seeds are a source of potent microproteinase inhibitors.
• The 'knottin' structure is crucial for the refolding and activity of these peptides.
• Synthetic strategies allow for the development of peptides with tailored inhibitory specificities.