Structure-guided protein engineering of human cathepsin L for efficient collagenolytic activity

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Structural Biology

Background:

• Protease specificity is crucial for biotechnological and therapeutic applications.
• Collagen degradation by collagenases is vital for extracellular matrix remodeling but implicated in pathologies when uncontrolled.
• Lysosomal cathepsin-K degrades collagen, while cathepsin-L lacks this ability.

Purpose of the Study:

• To engineer collagenolytic activity into cathepsin-L.
• To investigate the structural basis for collagen degradation by engineered cysteine cathepsins.

Main Methods:

• Systematic engineering of proline-specificity and glycosaminoglycans (GAG)-binding surface in cathepsin-L.
• Assessing collagenolytic activity of engineered mutants.
• Determining crystal structures of engineered mutants.
• Performing docking studies with substrates and GAG molecules.

Main Results:

• A proline-specific cathepsin-L mutant exhibited high specificity for prolyl-peptidic substrates but could not cleave collagen.
• Engineering a GAG-binding surface onto the proline-specific mutant enabled type-I collagen degradation in the presence of chondroitin-4-sulfate (C4-S).
• Crystal structures revealed key features of the engineered mutants, and docking studies identified structural determinants for collagenolysis.

Conclusions:

• Enzyme engineering can impart novel substrate specificity, such as collagen degradation, to proteases like cathepsin-L.
• The combination of proline-specificity and a GAG-binding surface is critical for achieving collagenolytic activity in engineered cysteine cathepsins.
• Understanding the structural basis of engineered collagenolysis provides insights for designing targeted proteases.