Lasso peptides are compact, bioactive peptides with topologically intriguing threaded structures. Experimental and computational studies suggest these unusual motifs may confer structure stabilization to boost protein function and biological activities. [1]. Microcin J25 (MccJ25) is an example of this class of peptide and is known to present bacterial RNA polymerase inhibition, being very effective against Gram-negative bacteria. [2] As an unexploited source of potential therapeutics, a general understanding of stable lasso peptide folding pathways would provide the basis to incorporate strategies to form lassos with new capacities and designable properties. Previous work on the folding of MccJ25 showed a folding pathway supporting the spontaneous formation of a left-handed pre-lasso conformer. However, natural MccJ25 adopts the right-handed chirality consistent with the topology of other natural lasso peptides. [3]
Our work aims to understand the spontaneous right-hand folding and characterize the likelihood and mechanism of pre-lasso folding of natural, synthetic, and designed peptides. In order to do that, unbiased Molecular Dynamics (MD) and Well-Tempered Metadynamics (WTMetaD) simulations were carried out using the latest all-atom AMBER ff19SB force field (FF). The concept to choose/build the collective variables (CVs) was to minimize the number of parameters that could accelerate the beta-hairpin formation and the turn that connects GLY1 with GLU8, forming the isopeptide bond of pre-tadpoles or pre-lassos.  To accomplish that, we applied Harmonic Linear Discriminant Analysis (HLDA) [4] on more than 500 possible descriptors from the structural ensembles of the unbiased simulations. We developed a triangulation-based algorithm to distinguish pre-lasso from pre-tadpole configurations based on the loop-tail distances. We also included a subroutine that identifies the chirality of a pre-lasso structure based on the arrangement of residues 1 to 8. Our exploratory MD simulations didn’t show the spontaneous formation of left-handed pre-lasso structures, only the presence of natural right-handed ones. We were able to generate 2D energy landscapes showing the distinct states pre-lasso (1% from the total clusters), pre-tadpole (3.5%), and pro-lasso (unfolded states), based on population distributions. Simulations using thioester auxiliaries at GLY1 and GLU8 residues show that these organic modifications increase the amount of pre-lasso (5%) and pre-tadpoles (6%). The results suggested the pre-lasso conformer is less stable than the pro-lasso, and transition barriers are markedly higher than 1.5 kcal/mol. Therefore, WTMetaD simulations are currently being performed to converge and quantify the free-energy differences and the energy barriers between the transitions.

Bioactive MccJ25 Lasso Peptide: Using Molecular Simulations to Understand its Folding Landscape

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