| Autor: |
Tan HN; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan., Liu WQ; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan., Ho J; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan., Chen YJ; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan., Shieh FJ; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan., Liao HT; Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu 300193, Taiwan., Wang SP; Institute of Biomedical Sciences, Academia Sinica, Taipei 115201, Taiwan., Hegemann JD; Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Saarland University Campus, 66123 Saarbrücken, Germany., Chang CY; Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu 300193, Taiwan., Chu J; Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan. |
| Abstrakt: |
Microcin J25 (MccJ25), a lasso peptide antibiotic with a unique structure that resembles the lariat knot, has been a topic of intense interest since its discovery in 1992. The precursor (McjA) contains a leader and a core segment. McjB is a protease activated upon binding to the leader, and McjC converts the core segment into the mature MccJ25. Previous studies suggested that these biosynthetic steps likely proceed in a (nearly) concerted fashion; however, there is only limited information regarding the structural and molecular intricacies of MccJ25 biosynthesis. To close this knowledge gap, we used AlphaFold2 to predict the structure of the precursor (McjA) in complex with its biosynthetic enzymes (McjB and McjC) and queried the critical predicted features by protein engineering. Based on the predicted structure, we designed protein variants to show that McjB can still be functional and form a proficient biosynthetic complex with McjC when its recognition and protease domains were circularly permutated or split into separate proteins. Specific residues important for McjA recognition were also identified, which permitted us to pinpoint a compensatory mutation (McjB M108T ) to restore McjA/McjB interaction that rescued an otherwise nearly nonproductive precursor variant (McjA T-2M ). Studies of McjA, McjB, and McjC have long been mired by them being extremely difficult to handle experimentally, and our results suggest that the AF2 predicted ternary complex structure may serve as a reasonable starting point for understanding MccJ25 biosynthesis. The prediction-validation workflow presented herein combined artificial intelligence and laboratory experiments constructively to gain new insights. |
