Lasso Peptides: Exploring the Folding Landscape of Nature’s Smallest Interlocked Motifs

Abstract

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

One Descriptor to Fold Them All: Harnessing Intuition and Machine Learning to Identify Transferable Lasso Peptide Reaction Coordinates

“…In our previous study, we discretized the conformational phase space of MccJ25 using a summation of the ring–tail distances combined with the distance between the isopeptide bond-forming residues, Gly1 and Glu8 . However, these descriptors were unable to fully distinguish the pre-lasso (Figure D) from the pre-tadpole (Figure E) conformation.…”

“…CVs can be valuable in the exploration and characterization of conformational phase space of biomolecules, especially for complex systems such as lasso peptides. , However, choosing appropriate CVs is not a trivial task, as different CVs may have different advantages and disadvantages depending on the system and the goal of the study. Herein, we explore two main approaches to CV selection: intuitive versus machine-learned.…”

“…However, revisiting the folding of MccJ25 with updated force fields recently demonstrated that it indeed forms the expected right-handed pre-lasso motif . The earlier observation of the left-handed pre-lasso conformation was attributed to the use of the GROMOS96 43A2 force field, which has since been refined to correct backbone dihedral parameters. − In addition, the recent simulations revealed that while the pre-lasso conformation of MccJ25 is metastable, the peptide readily transitions to the entropically favored unfolded and pre-tadpole conformations . This suggests that de novo pre-lasso folding is rare and that achieving the lasso motif synthetically will likely require enhanced stabilization of the pre-lasso conformation.…”

“…In our previous work, we identified parameters for discretizing the phase space of MccJ25 . However, an infrequent but non-negligible overlap in the pre-lasso and the pre-tadpole distributions was observed, rendering these parameters ineffective as CVs for distinguishing the two ensembles.…”

“…In this study, we attempt to identify differentiable CVs that distinguish the pre-lasso and pre-tadpole ensembles to characterize the folding landscape of lasso peptides. For model peptides, we choose MccJ25, ,, which is known for its unique β-hairpin secondary structure, as well as two shorter peptides lacking secondary structures, ulleungdin (Uln) and sungsanpin (Sun) . Once the isopeptide bonds are formed, each peptide retains the lasso scaffold due to a stopper residue (Tyr20, Tyr13, and Trp14 for MccJ25, Uln, and Sun, respectively) that prevents the tail from unthreading through the ring (Figure ).…”