Structure Prediction and Protein Engineering Yield New Insights into Microcin J25 Precursor Recognition.

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Microcin J25 (MccJ25) is a lasso peptide antibiotic discovered in 1992 with a unique lariat knot structure.
• Its biosynthesis involves a precursor (McjA) and two enzymes (McjB and McjC), but the intricate details remain largely unknown.

Purpose of the Study:

• To elucidate the structural and molecular mechanisms underlying MccJ25 biosynthesis.
• To leverage artificial intelligence for predicting complex protein interactions in antibiotic production.

Main Methods:

• Utilized AlphaFold2 to predict the ternary complex structure of McjA, McjB, and McjC.
• Employed protein engineering to validate critical predicted structural features and enzyme functions.
• Identified specific residues involved in McjA recognition by McjB.

Main Results:

• Confirmed McjB functionality and complex formation with McjC even with permuted or split domains, based on predicted structures.
• Identified key residues for McjA recognition and a compensatory mutation (McjB_M108T) that restored function to a precursor variant (McjA_T-2M).
• The predicted AlphaFold2 ternary complex structure serves as a viable model for studying MccJ25 biosynthesis.

Conclusions:

• The study presents a successful prediction-validation workflow combining AI and experimental methods to gain insights into MccJ25 biosynthesis.
• Demonstrated the plasticity of McjB's domains and identified critical interaction points, advancing the understanding of lasso peptide antibiotic production.