EXAMINING LEADER PEPTIDES AND FUSION PROTEINS OF LASSO PEPTIDES

Abstract

Lasso peptides are ribosomally synthesized and post-translationally modified peptides with a unique threaded lasso-like structure. Cyclization between the N-terminus and an acidic side chain forms a ring through which the C-terminal tail is threaded. The tail is prevented from slipping or “unthreading” by bulky amino acid side chains in the tail or by disulfide bonds between the ring and the tail. Their biosynthesis involves the enzymes B and C, which form the mature peptide by cleaving the lasso peptide leader peptide from the core peptide and cyclizing the core peptide, respectively. However, the role of the leader peptide and its relationship with the maturation enzymes is still unclear and not well understood. Therefore, the role of a highly conserved threonine residue in the penultimate position of the leader peptide was investigated in the lasso peptide, astexin-1, and its effect on peptide production was compared with similar studies done on other lasso peptides, microcin J25 and capistruin. Initial studies showed that only substitutions of similar size and shape of the threonine were tolerated. Astexin-1 was also found to be more sensitive to the changes in the leader peptide compared to microcin J25 and capistruin. Subsequently, to further evaluate the role of the penultimate threonine residue on the maturation enzymes, the recently engineered astexin-1 fusion protein was used to visualize the threonine’s effect on the B enzyme in the astexin-1 system. The results showed that the B enzyme was severely restricted by the threonine substitutions. In additional studies, capistruin was fused to an A1 leucine zipper with the same methods as the astexin-1 fusion protein to determine if lasso peptides with shorter tails would also be amenable to fusion. However, the observed inability of the peptide to be lassoed suggested the need and the importance of an extended linker region between the peptide and the fused protein for proper CapC enzyme function. The importance of the penultimate threonine residue in astexin-1, particularly its key role in the functioning of the B enzyme, has been demonstrated and should prove to be valuable in future lasso peptide biosynthesis studies. Moreover, the creation of the novel capistruin fusion protein reinforced the idea that the lasso peptide tail region could be utilized as a stable scaffold for protein engineering.