(102a) Maturation Mechanism of Microcin J25: Free Energy Analysis and Low-Dimensional Kinetics From Replica Exchange Molecular Dynamics Simulations

Microcin J25 (MccJ25) is a
21-residue naturally-occurring antibiotic peptide first isolated from fecal E.
coli [1]. MccJ25 is water-soluble and possesses potent bacteriocidal
activity at micromolar concentrations against certain gram negative bacteria,
with proven efficacy against strains of E. coli, Salmonella and Shigella
[1,2]. The primary bacteriocidal functionality is achieved via the inhibition
of transcription in the target prokaryote by blocking the secondary channel of
RNA polymerase [3]. MccJ25 forms a ?lassoed tail? structure in which the
N-terminus and Glu8 sidechain are linked by a peptide bond to form a lasso
through which the C-terminus is threaded and sterically locked in position by
bulky aromatic Phe19 and Tyr20 residues. This motif confers substantial
structural rigidity, rendering the peptide resistant to pH extremes (2 to 12),
8M urea or 10M guanidinium chloride, and autoclaving at 120°C for 15 minutes.
These characteristics make MccJ25 an attractive candidate as a novel antibiotic
or food preservative, and the lasso motif of interest as a constituent of
rationally designed peptides

Although the biosynthetic
production mechanism of MccJ25 is known and has been reconstructed in vitro
[4], the means by which the lasso structure is adopted is not well understood.
MccJ25 is synthesized both in vivo and in vitro by exposing the
58-residue linear pre-MccJ25 to two maturation enzymes [4]. Structural and
sequence homologies suggest that one enzyme is involved in cleaving off the 37
residue leader peptide and the other in catalyzing the formation of the lasso,
but the degree to which the enzymes are involved in assisting the peptide adopt
its native fold, as opposed to its spontaneous adoption, is unknown.

In this work, we have conducted
over 100ns of solvent-explicit replica exchange molecular dynamics (REMD)
simulations of the 21-residue linearized MccJ25 ? native MccJ25 in which the
peptide bond between the N-terminus and Glu8 sidechain has been hydrolyzed ? to
investigate the conformational ensemble explored by the linear peptide in the
absence of maturation enzymes. By constructing free energy landscapes parameterized
by the fraction of native contacts, we demonstrate that although the
conformational global free energy minimum corresponds to an ensemble of
structures containing ~70% of the native contacts, a local free energy well
corresponding to ~90% native contacts contains around 3% of the equilibrium
population. The structures in the latter well possess their N-terminal chain
looped around the C-terminal chain, placing the terminal nitrogen atom within
~1nm of the Glu8 delta carbon. These findings demonstrate that a significant
fraction the linearized MccJ25 spontaneously adopts native-like ?lassoed tail?
structures, suggesting that enzymatic assistance is not required in the
formation of the lasso. Furthermore, in the spirit of work by Yang et al. [5],
we analyze the short continuous segments of the REMD trajectories to
parameterize the drift and diffusion coefficients of a Fokker-Planck equation
describing the evolution of the probability distribution on the one-dimensional
free energy landscape. An associated Langevin equation is then used to produce
a statistically significant number of trajectories of the system to provide an
estimate of the time required for the linear peptide to reach the near-native
state from the global free energy minimum. Finally, we discuss extensions of
these analyses to higher-dimensional projections and the use of the diffusion
mapping technique [6] to systematically identify relevant order parameters.

1. Salomón, R.A.; Farías, R.N. Microcin J25, a Novel Antimicrobial Peptide Produced by Escherichia Coli. J. Bacteriol. 1992, 174, 7428-7435.
2. Semenova, E.; Yuzenkova, Y.; Peduzzi, J.; Rebuffat, S.; Severinov, K. Structure-Activity Analysis of Microcin J25: Distinct Parts of the Threaded Lasso Molecule are Responsible for Interaction with Bacterial RNA Polymerase. J. Bacteriol. 2005, 187, 3859-3863.
3. Mukhopadhyay, J.; Sineva, E.; Knight, J.; Levy, R.M.; Ebright, R.H. Antibacterial Peptide Microcin J25 Inhibits Transcription by Binding within and Obstructing the RNA Polymerase Secondary Channel. Mol. Cell 2004, 14, 739-751.
4. Rebuffat, S.; Blond, A.; Destoumieux-Garzón, D.; Goulard, C.; Peduzzi, J. Microcin J25, from Macrocyclic to the Lasso Structure: Implications for Biosynthetic, Evolutionary and Biotechnological Perspectives. Curr. Protein Pept. Sci. 2004, 5, 383-391.
5. Yang, S.; Onuchic, J.N.; García, A.E.; Levine, H. Folding Time Predictions from All-atom Replica Exchange Simulations. J. Mol. Biol. 2007, 372, 756-763.
6. Coifman, R.R.; Lafon, S.; Lee, A.B.; Maggioni, M.; Nadler, B.; Warner, F.; Zucker, S.W. Geometric Diffusions as a Tool for Harmonic Analysis and Structure Definition of Data: Diffusion Maps. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 21, 7426-7431.