Abstract

Lasso peptides are a class of ribosomally-synthesized and post-translationally modified peptides (RiPPs), characterized by their unique 1-rotaxane structure.1,2 Despite the relative simplicity of their biosynthetic gene cluster, our understanding of the lasso peptides biosynthesis is very limited. We infer that the lasso peptide maturation machinery is multiple enzymes involved, a precisely controlled process, however, most of the intermediates are still not able to be trapped and structurally analyzed. We use microcin J25 (MccJ25) as our project model, in which McjB and McjC proteins are the maturation enzymes working on the precursor peptide. We successfully expressed and purified MBP-tagged McjB protein and His-tagged McjC protein with different E. coli strains. MccJ25 was readily formed from a reconstitution assay using chemically synthesized McjA, MBP-McjB and His-McjC, proving that both the maturation proteins are active. A small amount of linear precursor was formed as an aberrant hydrolytic product, which has been confirmed to be due to the excessive MBP-McjB fusion protein in the assay. Product inhibition is also observed in the assay, suggesting that MccJ25 needs the ABC transporter, called McjD, to shuttle it outside cell membrane. Binding interaction between McjA, McjB and McjC are tested using isothermal titration calorimetry (ITC), McjA has a high binding affinity for MBP-McjB with KD = 0.12 μM, while it doesn’t show any affinity for the MBP control protein. Meanwhile, McjC is found to bind MBP-McjB with KD = 27 nM, whereas its interaction with the control MBP protein is almost nonexistent. 

Acknowledgement

This work was supported by the Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) SMSEGL20SC01.

Reference:

1. Maksimov, M.O.; Pan, S. J.; Link, A. J. Nat. Prod. Rep, 2014, 29, 996–1006.
2. Hegemann, J.D.; Zimmermann, M.; Xie, X.; Marahiel, M. Acc. Chem. Res, 2015, 48, 1909-1919.