Structural Dynamics of the Box C/D RNA Kink-Turn and Its Complex with Proteins: The Role of the A-Minor 0 Interaction, Long-Residency Water Bridges, and Structural Ion-Binding Sites Revealed by Molecular Simulations

Abstract

Kink-turns (K-turns) are recurrent elbow-like RNA motifs that participate in protein-assisted RNA folding and contribute to RNA dynamics. We carried out a set of molecular dynamics (MD) simulations using parm99 and parmbsc0 force fields to investigate structural dynamics of the box C/D RNA and its complexes with two proteins: native archaeal L7ae protein and human 15.5 kDa protein, originally bound to very similar structure of U4 snRNA. The box C/D RNA forms K-turn with A-minor 0 tertiary interaction between its canonical (C) and noncanonical (NC) stems. The local K-turn architecture is thus different from the previously studied ribosomal K-turns 38 and 42 having A-minor I interaction. The simulations reveal visible structural dynamics of this tertiary interaction involving altogether six substates which substantially contribute to the elbow-like flexibility of the K-turn. The interaction can even temporarily shift to the A-minor I type pattern; however, this is associated with distortion of the G/A base pair in the NC-stem of the K-turn. The simulations show reduction of the K-turn flexibility upon protein binding. The protein interacts with the apex of the K-turn and with the NC-stem. The protein-RNA interface includes long-residency hydration sites. We have also found long-residency hydration sites and major ion-binding sites associated with the K-turn itself. The overall topology of the K-turn remains stable in all simulations. However, in simulations of free K-turn, we observed instability of the key C16(O2')-A7(N1) H-bond, which is a signature interaction of K-turns and which was visibly more stable in simulations of K-turns possessing A-minor I interaction. It may reflect either some imbalance of the force field or it may be a correct indication of early stages of unfolding since this K-turn requires protein binding for its stabilization. Interestingly, the 16(O2')-7(N1) H- bond is usually not fully lost since it is replaced by a water bridge with a tightly bound water, which is adenine-specific similarly as the original interaction. The 16(O2')-7(N1) H-bond is stabilized by protein binding. The stabilizing effect is more visible with the human 15.5 kDa protein, which is attributed to valine to arginine substitution in the binding site. The behavior of the A-minor interaction is force-field-dependent because the parmbsc0 force field attenuates the A-minor fluctuations compared to parm99 simulations. Behavior of other regions of the box C/D RNA is not sensitive to the force field choice. Simulation with net-neutralizing Na(+) and 0.2 M excess salt conditions appear in all aspects equivalent. The simulations show loss of a hairpin tetraloop, which is not part of the K-turn. This was attributed to force field limitations.

Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations

“…In trajectory 3, which has the most significant principal movement, this angle difference is up to B351 (data not shown). This is consistent with the previous reports 40,43,89 that the larger and more flexible angle in apo-sRNA confirmed bound sRNA with the stabilization upon L7Ae binding.…”

“…Previous MD simulations have confirmed this flexibility. 43 In our study, principal component analysis (PCA) [63][64][65][66] was employed to discover the principal movements of these two stems. The ptraj module of AMBER11 was used to solve eigenvalues and corresponding eigenvectors from a covariance matrix and to calculate the contribution of each principal component.…”

“…This suggests that bound sRNA has a higher stability than the apo-structure. Spackova et al 43 discussed the choice of force fields in MD simulations. It could not be properly described by ff99SB/ff99bsc0 either with the A12 flipped to the vertical orientation or with these 4 nucleotides totally disrupted.…”

“…Several MD simulations of apo-sRNA or their complex have been made to reveal the recognition mechanism and the conformational change, [38][39][40] which also indicated that the flexibility of the V-shaped K-turn sRNA and the tertiary interactions between the sRNA and its chaperon protein play critical roles in the binding procedure. 38,39,[41][42][43] Among these previous observations, most attention was focused on the interactions between sRNA and its chaperon, while the internal folding kinetics of sRNA upon L7Ae-binding were poorly understood. In the current study, we compared the hinge motion and folding kinetics of L7Ae-bound and apo sRNA to further discover the mechanism of recognition between sRNA and L7Ae using MD simulations.…”

It's a loop world – single strands in RNA as structural and functional elements

“…It contains a similar sharp bend as observed in kink-turns, albeit with a curvature of approximately 908 and into the opposite direction, leading to a juxtaposition of the major grooves. The different known conformational states of kinkturns make them interesting targets for molecular dynamics studies we.g., (49)x. A possible explanation partially supported by fluorescence studies is that internal loops following the kink-turn consensus exist in a dynamic 3-state equilibrium (unbent state, kinked, reverse-kinked) (48).…”