(467i) Universal Folding Mechanisms of Lasso Peptides

Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique knotted structure. The threaded structure endows lasso peptides with remarkable thermal and proteolytic stability. However, understanding the universal folding process that leads to this lariat knot-like structure has remained a significant challenge in understanding the biosynthetic mechanism of its formation. To tackle this challenge, we have conducted 4 ms of aggregate atomic-level molecular dynamic simulations for 20 different structurally characterized lasso peptides. Our simulation results revealed two distinct types of folding mechanism of lasso peptides: flat folding landscape and uphill folding landscape. Our results show that the population of the pre-folded state is low for all lasso peptides. The flat folding landscapes are dominated by entropy such that the transition between pre-folded and unfolded state are readily observed but the residence time of the peptide in the folded state remains low. In the uphill folding landscape, a large free energy barrier separates the pre-folded and unfolded states. These results indicate that decreasing the entropy of the folded state (flat free energy landscape) and increasing the enthalpic interactions (uphill folding landscape) of the lasso preptides could facilitate the formation of the lasso structure in solution. We employ a deep learning architec- ture to cluster lasso folding pathways into distinct metastable path channels, which efficiently identifies the distinct folding mechanisms in solution. Furthermore, using out-of-distribution AI methods, we show that the formation of secondary structure is an essential feature of the transition state ensemble during lasso folding. To stabilize the transition state in the uphill folding landscape, we introduce mutations that enhance the folding of the microcin J25. We observed a strong correlation between simulation predicted beta-sheet content and lasso peptide production tested using cell-free biosynthesis. In summary, these findings provide insights into the folding mechanism of all natural lasso peptides with available structures and provide avenues for engineering the folding of lasso peptides in solution.