Crystallization, data collection and processing of the chymotrypsin-BTCI-trypsin ternary complex

Area of Science:

• Biochemistry
• Structural Biology
• Crystallography

Background:

• The black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a protein isolated from Vigna unguiculata seeds.
• BTCI is capable of simultaneously inhibiting both trypsin and chymotrypsin by forming a stable ternary complex.

Purpose of the Study:

• To crystallize and determine the X-ray crystal structure of the ternary complex formed between BTCI, trypsin, and chymotrypsin.
• To elucidate the structural basis of BTCI's dual inhibitory activity.

Main Methods:

• Crystallization using the sitting-drop vapour-diffusion method with specific precipitant solutions (HEPES, polyethylene glycol 6000, 2-methyl-2,4-pentanediol).
• X-ray diffraction data collection from a single crystal under cryogenic conditions to 2.7 Å resolution.
• Structure solution via molecular replacement using existing crystal structures of BTCI-trypsin binary complex and chymotrypsin.

Main Results:

• A stable ternary complex of trypsin, chymotrypsin, and BTCI was successfully crystallized.
• High-resolution X-ray diffraction data were obtained, enabling structural determination.
• The crystal structure of the ternary complex was solved, providing insights into the interaction.

Conclusions:

• The study successfully determined the crystal structure of the trypsin-BTCI-chymotrypsin ternary complex.
• This structural information provides a molecular understanding of how BTCI inhibits two distinct proteases simultaneously.
• The findings contribute to the understanding of protease-inhibitor interactions and protein complex formation.