Calpastatin simultaneously binds four calpains with different kinetic constants

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Calpastatin is the specific endogenous inhibitor of calpains, a family of calcium-dependent proteases.
• Calpain activity is implicated in various physiological and pathological processes.

Purpose of the Study:

• To investigate the calcium-dependent binding interaction between calpastatin and a calpain mutant without proteolysis.
• To characterize the binding kinetics of individual calpastatin inhibitory domains to calpain.

Main Methods:

• Surface plasmon resonance (SPR) was employed to study the enzyme-inhibitor interaction.
• An active site knock-out mutant of m-calpain was used to prevent proteolysis during binding studies.
• Individual inhibitory domains of calpastatin (CAST1-4) were expressed and purified for kinetic analysis.

Main Results:

• Calpastatin demonstrated the capacity to bind up to four molecules of calpain simultaneously.
• The dissociation constants (K(d)) for the interaction of CAST1, CAST2, CAST3, and CAST4 with calpain varied from picomolar to nanomolar.
• The order of affinity was CAST1 > CAST4 > CAST3 > CAST2, with similar association rates (k(on)) but vastly different dissociation rates (k(off)).

Conclusions:

• The differential affinity of calpastatin domains for calpain is primarily determined by their distinct dissociation rates (k(off)).
• Understanding these domain-specific interactions provides insights into the regulatory mechanisms of calpain activity.