The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.

The hairpin ribozyme is a prominent member of the group of small catalytic RNAs (RNA enzymes or ribozymes) because it does not require metal ions to achieve catalysis. Biochemical and structural data have implicated guanine 8 (G8) and adenine 38 (A38) as catalytic participants in cleavage and ligation catalyzed by the hairpin ribozyme, yet their exact role in catalysis remains disputed. To gain insight into dynamics in the active site of a minimal self-cleaving hairpin ribozyme, we have performed extensive classical, explicit-solvent molecular dynamics (MD) simulations on timescales of 50-150 ns. Starting from the available X-ray crystal structures, we investigated the structural impact of the protonation states of G8 and A38, and the inactivating A−1(2′-methoxy) substitution employed in crystallography. Our simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 profoundly distorts the active site. Thus MD simulations do not support a straightforward participation of the deprotonated G8 in catalysis. By comparison, the G8 enol tautomer is structurally well tolerated, causing only local rearrangements in the active site. Furthermore, a protonated A38H + is more consistent with the crystallography data than a canonical A38. The simulations thus support the notion that A38H + is the dominant form in the crystals, grown at pH 6. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the structures of active site and S-turn. Yet, we occasionally also observe formation of a stable A−1(2′-OH)…A38(N1) hydrogen bond, which documents the ability of the ribozyme to form this hydrogen bond, consistent with a potential role of A38 as general base catalyst. The presence of this hydrogen bond is, however, incompatible with the expected in-line attack angle necessary for self-cleavage, requiring a rapid transition of the deprotonated 2′-oxyanion to a position more favorable for in-line attack after proton transfer from A−1(2′-OH) to A38(N1). The simulations revealed a potential force field artifact, occasional but irreversible formation of 'ladder-like', underwound A-RNA structure in one of the external helices. Although it does not affect the catalytic center of the hairpin ribozyme, further studies are under way to better assess possible influence of such force field behavior on long RNA simulations.

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of [80 ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries of the three non-Watson-Crick basepairs that differ from the consensus bacterial sequence. The deep groove of Loop E motifs provides unique sites for cation binding. Binding of Mg 21 rigidifies Loop E and stabilizes its major groove at an intermediate width. In the absence of Mg 21 , the Loop E motifs show an unprecedented degree of inner-shell binding of monovalent cations that, in contrast to Mg 21 , penetrate into the most negative regions inside the deep groove. The spinach chloroplast Loop E shows a marked tendency to compress its deep groove compared with the bacterial consensus. Structures with a narrow deep groove essentially collapse around a string of Na 1 cations with long coordination times. The Loop E non-Watson-Crick basepairing is complemented by highly specific hydration sites ranging from water bridges to hydration pockets hosting 2 to 3 long-residing waters. The ordered hydration is intimately connected with RNA local conformational variations.

Ribozymes are catalytically competent examples of highly structured noncoding RNAs, which are ubiquitous in the processing and regulation of genetic information. Combining explicit-solvent molecular dynamics simulation and single molecule fluorescence spectroscopy approaches, we find that a ribozyme from a subviral plant pathogen exhibits a coupled hydrogen bonding network that communicates dynamic structural rearrangements throughout the catalytic core in response to site-specific chemical modification. Trapped long-residency water molecules are critical for this network and only occasionally exchange with bulk solvent as they pass through a breathing interdomain base stack. These highly structured water molecules line up in a string that may potentially also be involved in specific base catalysis. Our observations suggest important, still underappreciated roles for specifically bound water molecules in the structural dynamics and function of noncoding RNAs.coupled molecular motions ͉ hairpin ribozyme ͉ molecular dynamics ͉ proton wire ͉ specific base catalysis W ater is the universal solvent that supports all known forms of life. It is known to bind and stabilize the native structures of biopolymers such as RNA (1-10), but its precise role(s) in RNA function remain poorly understood. Highly structured noncoding (nc)RNAs, some endowed with catalytic functionality, have recently been recognized to outnumber protein-coding RNAs several-fold and to be of central importance in the processing and regulation of genetic information (11-13). Few techniques exist that can possibly provide insight into the intricate role(s) that water molecules play in the structurefunction relationships of this important class of biopolymers. We here have applied a combination of explicit-solvent molecular dynamics (MD) and single-molecule FRET (smFRET) approaches to reveal support for two distinct roles for water molecules in the function of a particularly compact catalytic ncRNA, the hairpin ribozyme, derived from a subviral plant pathogen (12).As is common for ncRNAs, the hairpin ribozyme relies on specific hydrogen bonding and base stacking to form an intricate tertiary structure with a solvent protected core (14). An interdomain Gϩ1:C25 Watson-Crick base pair reinforced by a Gϩ1:A38 stacking interaction, a 4-nt ribose zipper, and a specific hydrogen bonding pocket for an extruded U42 mediate docking of its domains A and B (Fig. 1a). smFRET studies have revealed the dynamic nature of these docking interactions (15-18), in which distal functional group modifications often significantly accelerate undocking. The latter observation led to the hypothesis that coupled molecular motions interconnect distal segments of the RNA (17). We here have confirmed this view by tracking the underlying hydrogen bonding network and finding that single water molecules trapped in the solvent protected catalytic core are integral components of this network.Despite intense efforts, the catalytic mechanism of the hairpin ribozyme is still ill-understood. F...

Explicit solvent molecular dynamics (MD) simulations were carried out for three RNA kissing-loop complexes. The theoretical structure of two base pairs (2 bp) complex of H3 stem-loop of Moloney murine leukemia virus agrees with the NMR structure with modest violations of few NMR restraints comparable to violations present in the NMR structure. In contrast to the NMR structure, however, MD shows relaxed intermolecular G-C base pairs. The core region of the kissing complex forms a cation-binding pocket with highly negative electrostatic potential. The pocket shows nanosecond-scale breathing motions coupled with oscillations of the whole molecule. Additional simulations were carried out for 6 bp kissing complexes of the DIS HIV-1 subtypes A and B. The simulated structures agree well with the X-ray data. The subtype B forms a novel four-base stack of bulged-out adenines. Both 6 bp kissing complexes have extended cation-binding pockets in their central parts. While the pocket of subtype A interacts with two hexacoordinated Mg2+ ions and one sodium ion, pocket of subtype B is filled with a string of three delocalized Na+ ions with residency times of individual cations 1-2 ns. The 6 bp complexes show breathing motions of the cation-binding pockets and loop major grooves.

Molecular dynamics (MD) simulations were employed to investigate the structure, dynamics, and local base-pair step deformability of the free 16S ribosomal helix 44 from Thermus thermophilus and of a canonical A-RNA double helix. While helix 44 is bent in the crystal structure of the small ribosomal subunit, the simulated helix 44 is intrinsically straight. It shows, however, substantial instantaneous bends that are isotropic. The spontaneous motions seen in simulations achieve large degrees of bending seen in the X-ray structure and would be entirely sufficient to allow the dynamics of the upper part of helix 44 evidenced by cryo-electron microscopic studies. Analysis of local base-pair step deformability reveals a patch of flexible steps in the upper part of helix 44 and in the area proximal to the bulge bases, suggesting that the upper part of helix 44 has enhanced flexibility. The simulations identify two conformational substates of the second bulge area (bottom part of the helix) with distinct base pairing. In agreement with nuclear magnetic resonance (NMR) and X-ray studies, a flipped out conformational substate of conserved 1492A is seen in the first bulge area. Molecular dynamics (MD) simulations reveal a number of reversible alpha-gamma backbone flips that correspond to transitions between two known A-RNA backbone families. The flipped substates do not cumulate along the trajectory and lead to a modest transient reduction of helical twist with no significant influence on the overall geometry of the duplexes. Despite their considerable flexibility, the simulated structures are very stable with no indication of substantial force field inaccuracies.

Reverse kink-turn is a recurrent elbow-like RNA building block occurring in the ribosome and in the group I intron. Its sequence signature almost matches that of the conventional kink-turn. However, the reverse and conventional kink-turns have opposite directions of bending. The reverse kink-turn lacks basically any tertiary interaction between its stems. We report unrestrained, explicit solvent molecular dynamics simulations of ribosomal and intron reverse kink-turns (54 simulations with 7.4 μs of data in total) with different variants (ff94, ff99, ff99bsc0, ff99χOL, and ff99bsc0χOL) of the Cornell et al. force field. We test several ion conditions and two water models. The simulations characterize the directional intrinsic flexibility of reverse kink-turns pertinent to their folded functional geometries. The reverse kink-turns are the most flexible RNA motifs studied so far by explicit solvent simulations which are capable at the present simulation time scale to spontaneously and reversibly sample a wide range of geometries from tightly kinked ones through flexible intermediates up to extended, unkinked structures. A possible biochemical role of the flexibility is discussed. Among the tested force fields, the latest χOL variant is essential to obtaining stable trajectories while all force field versions lacking the χ correction are prone to a swift degradation toward senseless ladder-like structures of stems, characterized by high-anti glycosidic torsions. The type of explicit water model affects the simulations considerably more than concentration and the type of ions.

The presence of Kink-turns (Kt) at key functional sites in the ribosome (e.g., A-site finger and L7/L12 stalk) suggests that some Kink-turns can confer flexibility on RNA protuberances that regulate the traversal of tRNAs during translocation. Explicit solvent molecular dynamics demonstrates that Kink-turns can act as flexible molecular elbows. Kink-turns are associated with a unique network of long-residency static and dynamical hydration sites that is intimately involved in modulating their conformational dynamics. An implicit solvent conformational search confirms the flexibility of Kink-turns around their X-ray geometries and identifies a second low-energy region with open structures that could correspond to Kink-turn geometries seen in solution experiments. An extended simulation of Kt-42 with the factor binding site (helices 43 and 44) shows that the local Kt-42 elbow-like motion fully propagates beyond the Kink-turn, and that there is no other comparably flexible site in this rRNA region. Kink-turns could mediate large-scale adjustments of distant RNA segments.