Characterization of caulonodin lasso peptides revealed unprecedented N-terminal residues and a precursor motif essential for peptide maturation

Abstract

Lasso peptides, a peculiar family of ribosomally assembled and post-translationally modified peptides (RiPPs), possess a fascinating 3D structure, which can confer rigidity and stability against chemical and thermal denaturation. Their distinctive “lariat knot” structure is accountable for their antibacterial, enzyme inhibitory and receptor antagonist activities. While the biosynthetic machinery was recently characterized, the rules concerning the formation of this unique lasso structure on the basis of their peptide sequences remain elusive. Restrictions such as the length of the peptide, the size of the ring, or the nature of the amino acids associated with the lasso fold stabilization were recently overhauled by the identification of new members of this RiPP family. In this work we demonstrate the isolation of four genome-mining-predicted lasso peptides featuring the unprecedented amino acids serine or alanine at position 1 of the core peptide. By a mutational approach we were able to predict the lasso fold for four peptides (caulonodins IV to VII). This prediction was confirmed for caulonodin V by the full elucidation of its 3D-structure via NMR and for caulonodin VI by the determination of long range NOE-contacts. Furthermore, the substrate specificity of the biosynthetic machinery for the atypical position 1 was probed. Additionally, utilizing the recent growth of functional lasso peptide precursor sequences we were able to identify a conserved motif in the C-terminal part of the leader peptide through bioinformatics analysis. Employing an extensive in vivo analysis for substitution tolerance of the biosynthetic machinery in this conserved region confirmed the significance of several residues, indicating that the predicted motif is very likely a general leader peptide recognition sequence specific for lasso peptide maturation.