(700e) Spontaneous Lasso Formation in Pro-Microcin J25: Replica Exchange Molecular Dynamics and Nonlinear Dimensionality Reduction

Ferguson, A. L., Princeton University
Debenedetti, P. G., Princeton University
Panagiotopoulos, A. Z., Princeton University

Microcin J25 (MccJ25) is a 21-residue antimicrobial ?lasso? peptide produced by E. coli, which disrupts RNA transcription in the prokaryotic target cell [1,2]. The structure of MccJ25 is a ?lassoed? β-hairpin in which the N-terminus wraps around the C-terminal strand in clockwise or ?right-handed? manner, and is sealed in place by an amide bond with the Glu8 side chain [3,4,5]. The C-terminus is sterically prohibited from slipping out by the aromatic rings borne by the Phe19 and Tyr20 ?steric locks? flanking the lasso. The lasso structure is very rigid, imparting remarkable resistance to protease degradation, and thermal and chaotropic stability: full activity is retained after autoclaving at 120°C for 15 minutes, exposure to 8M urea at 65°C for 16 hours or immersion in buffer solutions at pH 2-12 [1]. Interest has centered on this peptide as an antibiotic or food preservative, and as a useful protein-engineering motif.

The synthesis of active MccJ25 requires an 8-residue leader sequence and two maturation enzymes [6,7], but the precise functions of these entities are not well understood. In this work we perform molecular dynamics simulations of the 21-residue linear precursor of MccJ25, pro-MccJ25, to determine the conformational ensemble explored by the peptide in isolation, and thereby infer putative roles for the leader peptide and maturation enzymes.

We have conducted 120ns of replica exchange molecular dynamics (REMD) simulations of pro-MccJ25 in explicit solvent, and applied the diffusion map [8,9,10] as a nonlinear dimensionality reduction technique to systematically extract dynamically meaningful order parameters. Two global order parameters describing the identity of the residue at the β-turn position, and the value of the Glu8 Ψ angle, are identified as good descriptors of the fundamental dynamical motions of the peptide. Furthermore, we identify three distinct folding pathways associated with global hydrophobic collapse, adoption of an ?unwrapped? left-handed coil, and the spontaneous adoption of a left-handed lasso.

Targeted molecular dynamics simulations in the vicinity of the left-handed lasso, coupled with principal components analysis (PCA) and normal mode analysis (NMA), indicates that the lasso is formed by the lateral docking of the N-terminus behind the Phe19 steric lock. The low-entropy coil conformation is energetically stabilized by an electrostatic interaction between the positively charged N-terminal amino and negatively charged Glu8 carboxylate, mediated by a linear hydrogen bond network comprising the Ser18 hydroxyl and two interstitial water molecules. Similar techniques applied in the vicinity of the closest approach to a right-handed lasso, suggest that approach of the N-terminus into such conformations is sterically blocked by the concerted motion of the Phe19 aromatic side chain around the χ1 torsional angle.

Remarkably, we find that pro-MccJ25 spontaneously adopts a left-handed lasso conformation, in contrast to the right-handed topology of native MccJ25. Our results suggest possible roles for the leader sequence and maturation enzymes in facilitating the adoption of right-handed lassos during MccJ25 biosynthesis.

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