Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation

Abstract

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post-translational modification of the precursor McjA by the ATP-dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three-dimensional folding of MccJ25. We further showed that the N-terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43-S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23-Q34 in the N-terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.

Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides

“…Recently, several groups have reported the in vitro mutagenesis of lasso peptides (Yan et al, 2012;Burkhart et al, 2015;Assrir et al, 2016;Zhu et al, 2016a;Cheung et al, 2016;DiCaprio et al, 2019;Koos and Link, 2019;Si et al, 2021). In a foundational study, the biosynthesis of MccJ25 was reconstituted in vitro by incubating the precursor peptide McjA with processing enzymes McjB and McjC in the presence of ATP and Mg 2+ (Yan et al, 2012).…”

Identification and classification of papain-like cysteine proteinases

“…DUF553 (PF04473) proteins catalyze post-translational modifications of proteins by transferring, for example, peptidyl glutamine residues to a variety of amines ( 117 ). C96 family (Transglut_core3, PF13471) includes Escherichia coli McjB, which proteolytically processes McjA into a mature antimicrobial factor ( 118 ).…”

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide

“…Worth noting is that a previous NMR study showed that both McjB and McjC bind to the N-terminus of the leader sequence and that McjC interacts predominantly with the V6-S18 region of the McjA core sequence. 144 Since this region largely defines the β-hairpin structure in MccJ25, it was proposed that McjC binding to the peptide may facilitate the pre-folding process. These data hint at the important roles of the C protein in substrate–protein interactions that remain to be discovered.…”