Mapping L1 Ligase Ribozyme Conformational Switch

Abstract

L1 Ligase (L1L)molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5’-to-3’ phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA World hypothesis. L1L crystal structure captures two distinct conformations that differ by a re-orientation of one of the stems by around 80 Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution, and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a 3-state/2-step process. The first step involves a large-amplitude swing that re-orients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network.

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

“…York and co-workers studied the mechanism of RNA backbone ligation catalyzed by the L1 ligase. 1297 The crystal structure of the L1 ligase revealed two crystallographically independent conformations within an asymmetric unit cell. 1298 The authors used MD simulations and enhanced sampling techniques to probe the conformational pathway for interconverting the two observed states.…”

“… 1298 The authors used MD simulations and enhanced sampling techniques to probe the conformational pathway for interconverting the two observed states. 1297 They found the interconversion to include two steps: a hinge-like motion of the C-stem, followed by allosteric activation of the active site. Thus, the crystallographically observed conformations may correspond to active and inactive forms of the ligase.…”

Native States of Fast-Folding Proteins Are Kinetic Traps

“…14–19 Using network analysis, with specific conformations represented by nodes, and the transitions between conformations represented by edges, we can visualize the entire accessible free energy landscape of a protein in a single network graph. 14,20–24 However, questions still remain as to the best way to generate these graphs, and how we should interpret their main features.…”

Multiscale Methods for Computational RNA Enzymology

“…The past few decades have witnessed significant maturation of molecular mechanical force fields for nucleic acids based on relatively simple fixed charge models and pairwise potentials for non-bonded interactions (Wang et al, 2000; Pérez et al, 2007; Zgarbová et al, 2011; Brooks et al, 2009). The computational efficiency of these models allows MD simulations to routinely access μs timescales (Salomon-Ferrer, Götz, Poole, Le Grand, & Walker, 2013; Dror, Dirks, Grossman, Xu, & Shaw, 2012), making it a viable method for capturing large scale conformational changes in catalytic riboswitches (Giambaşu et al, 2010; Giambaşu, Lee, Scott, & York, 2012). Taken together, these developments have provided insight into the condensed phase structure and dynamics of ribozymes both in their pre-cleaved ground state and at various points along a reaction path (T.-S. Lee, Giambaşu, Harris, & York, 2011; T.-S. Lee et al, 2010; T.-S. Lee, Wong, Giambasu, & York, 2013).…”