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 5' to 3' 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.

FIGURE 1.

(A) Ribbon representations of the crystallographic presumed inactive undocked (left) and active docked (right) conformations of the L1 ligase. Changes in four virtual torsions (labeled and colored) distinguish between the conformers. The regions that do not change significantly are shown in gray. (B) Variations of the virtual torsions (ΔTorsion, where Torsion indicates either η or θ) between the docked (active) and undocked (inactive) conformers as found in the crystal structure. The Δη (black) and Δθ (red) are shown. Only four virtual torsions, θ₁₉, θ₃₇, η₃₈, and η₄₄, show significant deviations (80.0° or more), whereas all other virtual torsions show relatively minor changes (typically, less than 25.0°). (C) Representative snapshots from the simulations illustrating the coupled on–off conformational switch–catalytic pathway starting from the active and inactive conformations, both in precursor and product states. (Middle panel) Representative snapshots from the simulations are shown with stems A and B (in yellow wire-frame surface) aligned and stem C in ribbons with transparent surfaces. (Left panel) Contacts important for stabilization of intermediate states that involve the conserved U₁₉ and stem B. (Right panel) Interaction patterns for three states observed in the simulations between G₁/GTP₁ and the noncanonically base-paired ligation site. The states that do not appear in the crystal structure are indicated with curly arrows.

FIGURE 2.

(A,B) The U₃₈ loop is responsible for allosteric control of the catalytic step by transitioning from a “closed” conformation, labeled “U conformation” (specific, as shown by our simulations, to the inactive/undocked conformers), to an “open” conformation, labeled “D conformation” (specific to the active/docked conformations). (A) Representative conformations for the U and D conformations. (B) Upper panel: During the Prod-D-UF simulation, where unfolding of the docked state was induced by removal of key interstem interactions, the complete transition between the two states was observed by monitoring θ₃₇ and η₃₈ virtual torsions. The values of the θ₃₇ and η₃₈ found in the two crystallized conformers are shown with blue and orange ×’s. (Lower panel) The θ₃₇/η₃₈ space sampled in the Prod-D-UF overlaps with regions sampled during the Prod-D (red) and Prod-U (blue) simulations. The center of each distribution is marked with black ×’s. For additional details on the distribution of the two virtual torsions during the Prod-D and Prod-U simulations relative to the Prod-D-UF simulation, see Supplemental Material. (C) Exploring connections between the flexibility of the three-way junction and the L1 ligase conformational switch. Representative structures for the conformational transition along θ₁₈ toward the active conformation that brings U₁₉ in the close proximity of stem B. Structure 1 is similar to the undocked crystal conformation (θ₁₈ ≈ −110°). Structure 2 is the transition structure between structures 1 and 3 (θ₁₈ ≈ 170°). Structure 3 shows a set of specific tertiary interactions between the conserved U₁₉ and stem B (θ₁₈ ≈ 90°). The time evolution of the hydrogen bonding contacts as well as the overall distribution of the virtual torsions can be found in Supplemental Material. (D) The noncanonically base-paired ligation site exhibits a high degree of conformational variability, passing through a series of three states (clusters 1, 2, and 3) characterized by specific hydrogen bond patterns between GTP₁/G₁ and the ligation site. Shown is the arrangement of the ligation site in the case of the Prec-MgTP simulations. (E) The time evolution of probabilities of the three cluster states, and their correlation with the formation of specific U₇₁:H_O3′–GTP₁:O_2Pα interactions, are shown for the Prec-D-MgTP simulation. For the corresponding Prec-D-XTP simulation results, see Supplemental Material.