Investigation of Protein-Protein Interactions in Lasso Peptide Biosynthesis and the Discovery of New Antimicrobial Lasso Peptides

Abstract

Lasso peptides are a class of natural products defined by their unique threaded-ring topology. This highly constrained structure can confer increased stability against degradation by heat and by proteases. They have a diverse range of activities, from acting as receptor antagonists to exerting narrow spectrum antimicrobial effects. While the first few interesting lasso peptides were found through activity-guided efforts in the early 1990s, the advent of low cost genome sequencing has greatly increased our ability to discover new lasso peptides and understand the diversity in their biosynthetic gene clusters (BGCs).

One of the first variations seen in lasso peptide BGCs is that instead of just having two maturation enzymes, B and C, some BGCs had the B gene split into two open reading frames, B1 and B2. The function of B2 was predicted to be a cysteine protease, just like the B enzyme, but the function of B1 was unknown in 2014. In Chapter 2, in vitro studies were done to study the function of B1 using the model system for lariatin, an anti-mycobacterial lasso peptide that has the split-B architecture. We determined that LarB1 binds to the precursor peptide LarA with high affinity, and used peptide-mapping experiments to generate a model of the bound complex.

Chapters 3-5 are focused on the discovery and characterization of three new lasso peptides identified through genome mining. Chapters 3 and 4 focus on antimicrobial lasso peptides citrocin and ubonodin respectively, while Chapter 5 focuses on a structurally unique lasso peptide named pandonodin. All three peptides were heterologously expressed in Escherichia coli with good yield, and purified peptides were used for structure, activity, and biophysical characterization studies. In Chapter 3, we present the aqueous structure of citrocin, its antimicrobial activity against a few Enterobacteriaceae strains, its mechanism-of-action, and clues to its uptake mechanism into target strains. In Chapter 4, we present the aqueous structure of ubonodin, its potent antimicrobial activity against pathogenic Burkholderia strains, its mechanism-of-action, and some structure-activity studies. Finally, in Chapter 5, we present the structure of pandonodin in methanol, the largest lasso peptide discovered to date with surprising biophysical properties.