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RNA hydration: three nanoseconds of multiple molecular dynamics simulations of the solvated tRNAAsp anticodon hairpin1

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Abstract

The hydration of the tRNAAsp anticodon hairpin was investigated through the analysis of six 500 ps multiple molecular dynamics (MMD) trajectories generated by using the particle mesh Ewald method for the treatment of the long-range electrostatic interactions. Although similar in their dynamical characteristics, these six trajectories display different local hydration patterns reflecting the landscape of the “theoretical” conformational space being explored. The statistical view gained through the MMD strategy allowed us to characterize the hydration patterns around important RNA structural motifs such as a G-U base-pair, the anticodon U-turn, and two modified bases: pseudouridine and 1-methylguanine. The binding of ammonium counterions to the hairpin has also been investigated. No long-lived hydrogen bond between water and a 2′-hydroxyl has been observed. Water molecules with long-residence times are found bridging adjacent pro-Rp phosphate atoms. The conformation of the pseudouridine is stiffened by a water-mediated base-backbone interaction and the 1-methylguanine is additionally stabilized by long-lived hydration patterns. Such long-lived hydration patterns are essential to ensure the structural integrity of this hairpin motif. Consequently, our simulations confirm the conclusion reached from an analysis of X-ray crystal structures according to which water molecules form an integral part of nucleic acid structure. The fact that the same conclusion is reached from a static and a dynamic point of view suggests that RNA and water together constitute the biologically relevant functional entity.

Introduction

Water participates in all biochemical processes implying folding, recognition or catalysis of RNA molecules. Extensive surveys of crystallographic data have led to the view that water is an integral part of nucleic acid structures Westhof 1988, Westhof 1993, Westhof and Beveridge 1990, Schneider et al 1992. In helical duplexes, owing to the periodicity of the contacts between water and each repeating unit, specific hydration patterns are frequently observed. For example, a recent analysis of the high-resolution crystal structure of the regular r(CCCCGGGG) RNA duplex reveals a repetitive arrangement of water molecules around base-pairs and along the backbone (Egli et al., 1996). However, for non-periodic structures like single-stranded and loop regions, our knowledge of hydration motifs is scantier because of the limited number and the low resolution of the available crystallographic data. Besides, X-ray experiments give only partial views of the structure and dynamics of the hydration shell around nucleic acids (for discussion, see Westhof 1987, Westhof 1988). It is the hope that careful theoretical simulations on crystallographically well-described structures may fill in these information gaps.

The 17 nucleotide long tRNAAsp anticodon hairpin (Figure 1) contains both a helical and a non-helical component with structural motifs recurrent in many RNAs such as a wobble G-U base-pair (G30-U40) and a U-turn. Two modified nucleotides (Ψ32 and m1G37) are present in the loop. The wobble G-U base-pair is frequently associated with specific recognition between RNA and proteins (e.g. see Gabriel et al., 1996) or between RNAs (e.g. see Michel & Westhof, 1990). The peculiar hydration patterns around wobble G-U base-pairs and modified bases had been noted in crystallographic analyses (Westhof et al., 1988). However, the biological significance was difficult to assess and a conservative understanding was adopted; namely, that the recurrent and specific hydration sites map the favoured binding sites for the polar atoms of a potential partner in a complex.

In order to investigate the role of water in the stabilization of specific structural motifs and in the function of modified nucleotides in RNA, we have analyzed a set of six 500 ps MD simulations (S1 to S6) of the fully hydrated (1143 water molecules) and neutralized (16 NH4+ counterions) tRNAAsp anticodon hairpin. These trajectories were generated by using the particle mesh Ewald (PME) method Darden et al 1993, Essmann et al 1995 for the evaluation of the long-range electrostatic interactions together with the multiple molecular dynamics strategy (MMD) for an efficient sampling of the “theoretical” conformational space in the vicinity of the starting crystallographic structure (Auffinger et al., 1995). The PME method allows for the generation of stable nucleic acid trajectories on the nanosecond time-scale Cheatham et al 1995, York et al 1995, Louise-May et al 1996 while removing the artefacts commonly associated with the use of classical truncation schemes known to affect severely the stability and the dynamics of the simulated systems Smith and Pettitt 1991, Auffinger and Beveridge 1995. The MMD method, which consists of generating different uncorrelated trajectories by slightly perturbing the initial velocities, has proven to be useful for the evaluation of the stability of several simulation protocols Auffinger et al 1995, Auffinger et al 1996. From a previous analysis of the same set of six MD simulations, the dynamical behavior of the intramolecular tertiary interactions structuring the tRNAAsp anticodon hairpin has been extensively described (Auffinger & Westhof, 1996). The quality of these simulations allowed us to demonstrate, for the first time by molecular dynamics simulations, the stabilizing contributions of C-H…O interactions in RNA structures. Further, from the same set of simulations as well as from a 500 ps simulation of the full tRNAAsp molecule, we focused on the hydration of C-H groups (Auffinger et al., 1997). It was concluded that, in agreement with the results of X-ray studies, acidic C-H groups interact with water molecules via H-bonds thereby contributing to the enthalpic stabilization of specific hydration patterns.

Here, we focus on the hydration of hydrophilic atoms of the anticodon hairpin and more specifically on the following topics: (1) the hydration of the 2′-hydroxyl groups, which constitutes the sole chemical difference between RNA and DNA; (2) the hydration of the G-U base-pair; (3) the structural role of modified nucleotides; and (4) the structural significance of water molecules displaying long-residence times such as water molecules bridging particular solute atoms. We further address issues relative to the sampling of the conformational space during MD and to the use of the MMD strategy.

Section snippets

Results and discussion

The tRNAAsp anticodon hairpin contains a variety of hydrogen bond donor (ribose HO2′; base (G)H1/H21/H22, (C)H41/H42, (U)H3, (Ψ)H1/H3, (m1G)H21/H22) and acceptor sites (backbone O3′, OR, OS, O5′; ribose O2′, O4′; base (G)N3/N7/O6, (C)N3/O2, (U)O2/O4, (Ψ)O2/O4, (m1G)N3/N7/O6; see Figure 2). Among them, hydroxyl, imino and amino hydrogen atoms as well as non-protonated N3 and N7 base atoms may establish one hydrogen bond contact with the solvent, and oxygen atoms may form simultaneously up to two

Acknowledgements

The authors thank Dr Thomas Hermann for stimulating discussions and critical comments on the manuscript. P.A. thanks the program CM2AO from ORGANIBIO (28, rue Saint Dominique, Paris, France) and the “Fondation pour la Recherche Médicale” for support. The authors acknowledge the IDRIS computing center, which provided computer time, the Peter Kollman group (UCSF), which provided the latest version of the MD package used for this study, as well as Georges Wipff and Etienne Engler for the use of

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