Local binding with globally distributed changes in a small protease inhibitor upon enzyme binding

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• Serine protease inhibitors are crucial regulators of biological processes.
• The pacifastin inhibitor family, including SGCI, plays a role in insect immunity.
• Understanding inhibitor-enzyme interactions is key to drug development.

Purpose of the Study:

• To investigate the complexation of SGCI with bovine chymotrypsin using NMR spectroscopy.
• To elucidate the structural and dynamic changes in SGCI upon binding to chymotrypsin.
• To challenge the classical 'scaffold + binding loop' model for serine protease inhibitors.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy, specifically (1)H-(15)N HSQC.
• Titration of SGCI with increasing amounts of bovine chymotrypsin.
• Analysis of chemical shift perturbations and dynamic changes.

Main Results:

• Demonstrated tight and specific binding between SGCI and bovine chymotrypsin.
• Observed significant structural and dynamic changes in SGCI, extending beyond the canonical binding loop.
• Identified retention of some dynamic features of the free inhibitor in the complex.
• Concluded that minor structural transitions explain most, but not all, observed chemical shift changes.

Conclusions:

• The classical 'scaffold + binding loop' model may be insufficient for describing pacifastin inhibitor interactions.
• SGCI's interaction with chymotrypsin involves long-range effects, suggesting a more complex binding mechanism.
• This unique binding mode could explain the evolution of both specific and non-specific inhibitors within this family.