Substrate interactions guide cyclase engineering and lasso peptide diversification

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Lasso peptides are stable molecules with a unique rotaxane conformation.
• The mechanism of ATP-dependent lasso cyclase in forming these peptides is poorly understood due to enzyme instability.
• Understanding this process is key to harnessing lasso peptides for therapeutic applications.

Purpose of the Study:

• To elucidate the mechanism by which lasso cyclases constrain substrate peptides into lasso peptide products.
• To engineer lasso cyclases for novel substrate acceptance and therapeutic applications.

Main Methods:

• Combined substrate tolerance data, structural predictions, and bioinformatic analysis.
• Utilized molecular dynamics simulations and mutational scanning to model enzyme-substrate interactions.
• Rationally engineered diverse lasso cyclases to alter substrate specificity.

Main Results:

• Developed a model for substrate peptide orientation within the lasso cyclase active site.
• Successfully engineered multiple lasso cyclases to accept previously rejected substrates.
• Demonstrated robust production of novel lasso peptide variants with high affinity for integrin αvβ8.

Conclusions:

• The study provides a mechanistic model for lasso cyclase activity.
• Enzyme engineering successfully expanded substrate specificity and created novel therapeutic candidates.
• Engineered lasso cyclases offer a promising platform for developing anti-cancer agents targeting integrin αvβ8.