Structure Prediction\nand Protein Engineering Yield\nNew Insights into Microcin J25 Precursor Recognition

Microcin J25 (MccJ25),\na lasso peptide antibiotic with a unique\nstructure that resembles the lariat knot, has been a topic of intense\ninterest since its discovery in 1992. The precursor (McjA) contains\na leader and a core segment. McjB is a protease activated upon binding\nto the leader, and McjC converts the core segment into the mature\nMccJ25. Previous studies suggested that these biosynthetic steps likely\nproceed in a (nearly) concerted fashion; however, there is only limited\ninformation regarding the structural and molecular intricacies of\nMccJ25 biosynthesis. To close this knowledge gap, we used AlphaFold2\nto predict the structure of the precursor (McjA) in complex with its\nbiosynthetic enzymes (McjB and McjC) and queried the critical predicted\nfeatures by protein engineering. Based on the predicted structure,\nwe designed protein variants to show that McjB can still be functional\nand form a proficient biosynthetic complex with McjC when its recognition\nand protease domains were circularly permutated or split into separate\nproteins. Specific residues important for McjA recognition were also\nidentified, which permitted us to pinpoint a compensatory mutation\n(McjB<sub>M108T</sub>) to restore McjA/McjB interaction that rescued\nan otherwise nearly nonproductive precursor variant (McjA<sub>T–2M</sub>). Studies of McjA, McjB, and McjC have long been mired by them being\nextremely difficult to handle experimentally, and our results suggest\nthat the AF2 predicted ternary complex structure may serve as a reasonable\nstarting point for understanding MccJ25 biosynthesis. The prediction-validation\nworkflow presented herein combined artificial intelligence and laboratory\nexperiments constructively to gain new insights.