Identification of dynamical hinge points of the L1 ligase molecular switch

Abstract

The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 59 to 39 phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive-active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.

Mapping L1 Ligase Ribozyme Conformational Switch

“…The two conformations differ by a re-orientation of stem C tip by ˜80 Å . Based on analysis of the hydrogen-bonding patterns between the active site and substrate, we have previously shown [48] how the dynamics of the active site exhibits two completely different patterns of conformational variation in the active and inactive forms. More precisely, the catalytic site was shown to span three conformational states in its active form, whereas the dynamics of the inactive form was unimodal.…”

“…A minimal set of virtual torsions was identified to be sufficient to differentiate between the two conformers. [48] Based on the presence/absence of specific contacts in the ligation site and evolutionarily conserved regions of the bulged loop situated on stem C, it was proposed that the conformers represented catalytically active “ on ” and inactive “ off ” states. [105] We will refer to these two crystallized structures as active/docked and inactive/undocked, respectively.…”

“…In our previous analysis of the L1L structure and dynamics [48]we identified a set of dynamical hinge points using a series of molecular dynamics (MD) simulations of the precursor and product states that captured fluctuations around the active and inactive conformers. The hinge points were located in highly evolutionarily conserved regions of L1L sequence and were identified based on analysis of the distribution of virtual torsions.…”

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).…”

A new way to see RNA

“…In the meantime, the η/θ formalism was used as a platform for the development and use of new tools designed to explore the diversity of RNA conformational space (Beuth et al . 2005; Correll & Swinger, 2003; Giambasu et al . 2010; Huppler et al .…”