rRNA C-Loops: Mechanical Properties of a Recurrent Structural Motif.

C-loop is an internal loop motif found in the ribosome and used in artificial nanostructures. While its geometry has been partially characterized, its mechanical properties remain elusive. Here we propose a method to evaluate global shape and stiffness of an internal loop. The loop is flanked by short A-RNA helices modeled as rigid bodies. Their relative rotation and displacement are fully described by six interhelical coordinates. The deformation energy of the loop is assumed to be a general quadratic function of the interhelical coordinates. The model parameters for isolated C-loops are inferred from unrestrained all-atom molecular dynamics simulations. C-loops exhibit high twist as reported earlier, but also a bend and a lateral displacement of the flanking helices. Their bending stiffness and lateral displacement stiffness are nearly isotropic and similar to the control A-RNA duplexes. Nevertheless, we found systematic variations with the C-loop position in the ribosome and the organism of origin. The results characterize global properties of C-loops in the full six-dimensional interhelical space and enable one to choose an optimally stiff C-loop for use in a nanostructure. Our approach can be readily applied to other internal loops and extended to more complex structural motifs.

Simulations of A-RNA duplexes. The effect of sequence, solute force field, water model, and salt concentration.

We have carried out an extended reference set of explicit solvent molecular dynamics simulations (63 simulations with 8.4 µs of simulation data) of canonical A-RNA duplexes. Most of the simulations were done using the latest variant of the Cornell et al. AMBER RNA force field bsc0χ(OL3), while several other RNA force fields have been tested. The calculations show that the A-RNA helix compactness, described mainly by geometrical parameters inclination, base pair roll, and helical rise, is sequence-dependent. In the calculated set of structures, the inclination varies from 10° to 24°. On the basis of simulations with modified bases (inosine and 2,6-diaminopurine), we suggest that the sequence-dependence of purely canonical A-RNA double helix is caused by the steric shape of the base pairs, i.e., the van der Waals interactions. The electrostatic part of stacking does not appear to affect the A-RNA shape. Especially visible is the role of the minor groove amino group of purines. This resembles the so-called Dickerson-Calladine mechanical rules suggested three decades ago for the DNA double helices. We did not identify any long-living backbone substate in A-RNA double helices that would resemble, for example, the B-DNA BI/BII dynamics. The variability of the A-RNA compactness is due to mutual movements of the consecutive base pairs coupled with modest change of the glycosidic χ torsion. The simulations further show that the A-RNA compactness is modestly affected by the water model used, while the effect of ionic conditions, investigated in the range from net-neutral condition to ~0.8 M monovalent ion excess salt, is smaller.

A-minor tertiary interactions in RNA kink-turns. Molecular dynamics and quantum chemical analysis.

The RNA kink-turn is an important recurrent RNA motif, an internal loop with characteristic consensus sequence forming highly conserved three-dimensional structure. Functional arrangement of RNA kink-turns shows a sharp bend in the phosphodiester backbone. Among other signature interactions, kink-turns form A-minor interaction between their two stems. Most kink-turns possess extended A-minor I (A-I) interaction where adenine of the second A•G base pair of the NC-stem interacts with the first canonical pair of the C-stem (i.e., the receptor pair) via trans-sugar-edge/sugar-edge (tSS) and cis-sugar-edge/sugar-edge (cSS) interactions. The remaining kink-turns have less compact A-minor 0 (A-0) interaction with just one tSS contact. We show that kink-turns with A-I in ribosomal X-ray structures keep G═C receptor base pair during evolution while the inverted pair (C═G) is not realized. In contrast, kink-turns with A-0 in the observed structures alternate G═C and C═G base pairs in sequences. We carried out an extended set (~5 µs) of explicit-solvent molecular dynamics simulations of kink-turns to rationalize this structural/evolutionary pattern. The simulations were done using a net-neutral Na(+) cation atmosphere (with ~0.25 M cation concentration) supplemented by simulations with either excess salt KCl atmosphere or inclusion of Mg(2+). The results do not seem to depend on the treatment of ions. The simulations started with X-ray structures of several kink-turns while we tested the response of the simulated system to base substitutions, modest structural perturbations and constraints. The trends seen in the simulations reveal that the A-I/G═C arrangement is preferred over all three other structures. The A-I/C═G triple appears structurally entirely unstable, consistent with the covariation patterns seen during the evolution. The A-0 arrangements tend to shift toward the A-I pattern in simulations, which suggests that formation of the A-0 interaction is likely supported by the surrounding protein and RNA molecules. A-0 may also be stabilized by additional kink-turn nucleotides not belonging to the kink-turn consensus, as shown for the kink-turn from ribosomal Helix 15. Quantum-chemical calculations on all four A-minor triples suggest that there is a different balance of electrostatic and dispersion stabilization in the A-I/G═C and A-I/C═G triples, which may explain different behavior of these otherwise isosteric triples in the context of kink-turns.

Interaction of metal ions with biomolecular ligands: how accurate are calculated free energies associated with metal ion complexation?

To address fundamental questions in bioinorganic chemistry, such as metal ion selectivity, accurate computational protocols for both the gas-phase association of metal-ligand complexes and solvation/desolvation energies of the species involved are needed. In this work, we attempt to critically evaluate the performance of the ab initio and DFT electronic structure methods available and recent solvation models in calculations of the energetics associated with metal ion complexation. On the example of five model complexes ([M(II)(CH(3)S)(H(2)O)](+), [M(II)(H(2)O)(2)(H(2)S)(NH(3))](2+), [M(II)(CH(3)S)(NH(3))(H(2)O)(CH(3)COO)], [M(II)(H(2)O)(3)(SH)(CH(3)COO)(Im)], [M(II)(H(2)S)(H(2)O)(CH(3)COO)(PhOH)(Im)](+) in typical coordination geometries) and four metal ions (Fe(2+), Cu(2+), Zn(2+), and Cd(2+); representing open- and closed-shell and the first- and second-row transition metal elements), we provide reference values for the gas-phase complexation energies, as presumably obtained using the CCSD(T)/aug-cc-pVTZ method, and compare them with cheaper methods, such as DFT and RI-MP2, that can be used for large-scale calculations. We also discuss two possible definitions of interaction energies underlying the theoretically predicted metal-ion selectivity and the effect of geometry optimization on these values. Finally, popular solvation models, such as COSMO-RS and SMD, are used to demonstrate whether quantum chemical calculations can provide the overall free enthalpy (ΔG) changes in the range of the expected experimental values for the model complexes or match the experimental stability constants in the case of three complexes for which the experimental data exist. The data presented highlight several intricacies in the theoretical predictions of the experimental stability constants: the covalent character of some metal-ligand bonds (e.g., Cu(II)-thiolate) causing larger errors in the gas-phase complexation energies, inaccuracies in the treatment of solvation of the charged species, and difficulties in the definition of the reference state for Jahn-Teller unstable systems (e.g., [Cu(H(2)O)(6)](2+)). Although the agreement between the experimental (as derived from the stability constants) and calculated values is often within 5 kcal·mol(-1), in more complicated cases, it may exceed 15 kcal·mol(-1). Therefore, extreme caution must be exercised in assessing the subtle issues of metal ion selectivity quantitatively.

An RNA molecular switch: Intrinsic flexibility of 23S rRNA Helices 40 and 68 5'-UAA/5'-GAN internal loops studied by molecular dynamics methods.

Functional RNA molecules such as ribosomal RNAs frequently contain highly conserved internal loops with a 5'-UAA/5'-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair and finally by a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by MM-PBSA free energy calculations, locally enhanced sampling (LES) runs, targeted MD (TMD) and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na(+) vs. excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, loss of base-phosphate interaction and other instabilities. The alternative X-ray geometry even undergoes conformational transition towards the solution 2D structure. Free energy calculations confirm that the X-ray arrangement is less stable than the solution structure. LES, TMD and NEB provide a rather consistent pathway for interconversion between the X-ray and NMR structures. In simulations, the incomplete cWS A/U base pair of the NMR structure is water mediated and alternates with the canonical A-U base pair, which is not indicated by the NMR data. Completion of full cWS A/U base pair is prevented by the overall internal loop arrangement. In summary, the simulations confirm that the UAA/GAN internal loop is a molecular switch RNA module that adopts its functional geometry upon specific tertiary contexts.

Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome.

We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90-92 (H90-H92) of 23S rRNA; the 3WJ formed by H42-H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3'-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42-H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42-H97 contact.

An RNA Molecular Switch: Intrinsic Flexibility of 23S rRNA Helices 40 and 68 5'-UAA/5'-GAN Internal Loops Studied by Molecular Dynamics Methods.

Functional RNA molecules such as ribosomal RNAs (rRNAs) frequently contain highly conserved internal loops with a 5'-UAA/5'-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt a distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair, and finally a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by molecular mechanics, Poisson-Boltzmann, and surface area free energy calculations; locally enhanced sampling (LES) runs; targeted MD (TMD); and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na(+) versus excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, a loss of base-phosphate interaction, and other instabilities. The alternative X-ray geometry even undergoes a conformational transition toward the solution 2D structure. Free energy calculations confirm that the X-ray arrangement is less stable than the solution structure. LES, TMD, and NEB provide a rather consistent pathway for interconversion between the X-ray and NMR structures. In simulations, the incomplete cWS A/U base pair of the NMR structure is water-mediated and alternates with the canonical A-U base pair, which is not indicated by the NMR data. Completion of the full cWS A/U base pair is prevented by the overall internal loop arrangement. In summary, the simulations confirm that the UAA/GAN internal loop is a molecular switch RNA module that adopts its functional geometry upon specific tertiary contexts.

Dependence of A-RNA simulations on the choice of the force field and salt strength.

We present an extensive molecular dynamics study (0.6 micros in total) on three A-RNA duplexes. The dependence of the A-RNA geometry on force fields (Parm99 and Parmbsc0) and salt strength conditions (approximately 0.18 M net-neutralizing Na(+) and approximately 0.3 M KCl) was investigated. The Parmbsc0 force field makes the A-RNA duplex more compact in comparison to the Parm99 by preventing temporary alpha/gamma t/t flips common in Parm99 simulations. Nevertheless, since the alpha/gamma t/t sub-state occurs to certain extent in experimental A-RNA structures, we consider both force fields as viable. The stabilization of the A-RNA helices caused by the Parmbsc0 force field includes visible reduction of the major groove width, increase of the base pair roll, larger helical inclination and small increases of twist. Therefore, the Parmbsc0 shifts the simulated duplexes more deeply into the A-form. Further narrowing of the deep major groove is observed in excess salt simulations, again accompanied by larger roll, inclination and twist. The cumulative difference between Parm99/lower-salt and Parmbsc0/higher-salt simulations is approximately 4-8 A for the average PP distances, and -0.7 to -2.5 degrees, -2.0 to -5.4 degrees, -2.6 to -8.6 degrees and 1.7 to 7.0 degrees for the twist, roll, inclination and propeller, respectively. The effects of the force field and salt condition are sequence-dependent. Thus, the compactness of A-RNA is sensitive to the sequence and the salt strength which may, for example, modulate the end-to-end distance of the A-RNA helix. The simulations neatly reproduce the known base pair roll re-distribution in alternating purine-pyrimidine A-RNA helices.