Multiscale Modeling of Phosphate···π Contacts in RNA U-Turns Exposes Differences between Quantum-Chemical and AMBER Force Field Descriptions.

Phosphate···π, also called anion···π, contacts occur between nucleobases and anionic phosphate oxygens (OP2) in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). These contacts were investigated using state-of-the-art quantum-chemical methods (QM) to characterize their physicochemical properties and to serve as a reference to evaluate AMBER force field (AFF) performance. We found that phosphate···π interaction energies calculated with the AFF for dimethyl phosphate···nucleobase model systems are less stabilizing in comparison with double-hybrid DFT and that minimum contact distances are larger for all nucleobases. These distance stretches are also observed in large-scale AFF vs QM/MM computations and classical molecular dynamics (MD) simulations on several r(gcGNRAgc) tetraloop hairpins when compared to experimental data extracted from X-ray/cryo-EM structures (res. ≤ 2.5 Å) using the WebFR3D bioinformatic tool. MD simulations further revealed shifted OP2/nucleobase positions. We propose that discrepancies between the QM and AFF result from a combination of missing polarization in the AFF combined with too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms in addition to an exaggerated short-range repulsion of the r-12 LJ repulsive term. We compared these results with earlier data gathered on lone pair···π contacts in CpG Z-steps occurring in r(UNCG) tetraloops. In both instances, charge transfer calculations do not support any significant n → π* donation effects. We also investigated thiophosphate···π contacts that showed reduced stabilizing interaction energies when compared to phosphate···π contacts. Thus, we challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be explained by larger London dispersion.

Lone Pair π Contacts and Structure Signatures of r(UNCG) Tetraloops, Z-Turns, and Z-Steps: A WebFR3D Survey.

Z-DNA and Z-RNA have long appeared as oddities to nucleic acid scientists. However, their Z-step constituents are recurrently observed in all types of nucleic acid systems including ribosomes. Z-steps are NpN steps that are isostructural to Z-DNA CpG steps. Among their structural features, Z-steps are characterized by the presence of a lone pair…π contact that involves the stacking of the ribose O4' atom of the first nucleotide with the 3'-face of the second nucleotide. Recently, it has been documented that the CpG step of the ubiquitous r(UNCG) tetraloops is a Z-step. Accordingly, such r(UNCG) conformations were called Z-turns. It has also been recognized that an r(GAAA) tetraloop in appropriate conditions can shapeshift to an unusual Z-turn conformation embedding an ApA Z-step. In this report, we explore the multiplicity of RNA motifs based on Z-steps by using the WebFR3D tool to which we added functionalities to be able to retrieve motifs containing lone pair…π contacts. Many examples that underscore the diversity and universality of these motifs are provided as well as tutorial guidance on using WebFR3D. In addition, this study provides an extensive survey of crystallographic, cryo-EM, NMR, and molecular dynamics studies on r(UNCG) tetraloops with a critical view on how to conduct database searches and exploit their results.

Short-Range Imbalances in the AMBER Lennard-Jones Potential for (Deoxy)Ribose···Nucleobase Lone-Pair···π Contacts in Nucleic Acids.

The lone-pair···π (lp···π) (deoxy)ribose···nucleobase stacking is a recurring interaction in Z-DNA and RNAs that is characterized by sub-van der Waals lp···π contacts (<3.0 Å). It is a part of the structural signature of CpG Z-step motifs in Z-DNA and r(UNCG) tetraloops that are known to behave poorly in molecular dynamics (MD) simulations. Although the exact origin of the MD simulation issues remains unclear, a significant part of the problem might be due to an imbalanced description of nonbonded interactions, including the characteristic lp···π stacking. To gain insights into the links between lp···π stacking and MD, we present an in-depth comparison between accurate large-basis-set double-hybrid Kohn-Sham density functional theory calculations DSD-BLYP-D3/ma-def2-QZVPP (DHDF-D3) and data obtained with the nonbonded potential of the AMBER force field (AFF) for NpN Z-steps (N = G, A, C, and U). Among other differences, we found that the AFF overestimates the DHDF-D3 lp···π distances by ∼0.1-0.2 Å, while the deviation between the DHDF-D3 and AFF descriptions sharply increases in the short-range region of the interaction. Based on atom-in-molecule polarizabilities and symmetry-adapted perturbation theory analysis, we inferred that the DHDF-D3 versus AFF differences partly originate in identical nucleobase carbon atom Lennard-Jones (LJ) parameters despite the presence/absence of connected electron-withdrawing groups that lead to different effective volumes or vdW radii. Thus, to precisely model the very short CpG lp···π contact distances, we recommend revision of the nucleobase atom LJ parameters. Additionally, we suggest that the large discrepancy between DHDF-D3 and AFF short-range repulsive part of the interaction energy potential may significantly contribute to the poor performances of MD simulations of nucleic acid systems containing Z-steps. Understanding where, and if possible why, the point-charge-type effective potentials reach their limits is vital for developing next-generation FFs and for addressing specific issues in contemporary MD simulations.

Deflating the RNA Mg2+ bubble. Stereochemistry to the rescue!

Proper evaluation of the ionic structure of biomolecular systems through X ray and cryo-EM techniques remains challenging but is essential for advancing our understanding of the underlying structure/activity/solvent relationships. However, numerous studies overestimate the number of Mg2+ in deposited structures due to assignment errors finding their origin in improper consideration of stereochemical rules. Herein, to tackle such issues, we re-evaluate the PDBid 6QNR and 6SJ6 models of the ribosome ionic structure. We establish that stereochemical principles need to be carefully pondered when evaluating ion binding features, even when K+ anomalous signals are available as it is the case for the 6QNR PDB entry. For ribosomes, assignment errors can result in misleading conceptions of their solvent structure. For instance, present stereochemical analysis result in a significant decrease of the number of assigned Mg2+ in 6QNR, suggesting that K+ and not Mg2+ is the prevalent ion in the ribosome 1st solvation shell. We stress that the use of proper stereochemical guidelines in combination or not with other identification techniques, such as those pertaining to the detection of transition metals, of some anions and of K+ anomalous signals, is critical for deflating the current Mg2+ bubble witnessed in many ribosome and other RNA structures. We also stress that for the identification of lighter ions such as Mg2+, Na+, …, for which no anomalous signals can be detected, stereochemistry coupled with high resolution structures (<2.4 Å) remain the best currently available option.

Short but Weak: The Z-DNA Lone-Pair⋠⋠⋠π Conundrum Challenges Standard Carbon Van der Waals Radii.

Current interest in lone-pair⋅⋅⋅π (lp⋅⋅⋅π) interactions is gaining momentum in biochemistry and (supramolecular) chemistry. However, the physicochemical origin of the exceptionally short (ca. 2.8 Å) oxygen-to-nucleobase plane distances observed in prototypical Z-DNA CpG steps remains unclear. High-level quantum mechanical calculations, including SAPT2+3 interaction energy decompositions, demonstrate that lp⋅⋅⋅π contacts do not result from n→π* orbital overlaps but from weak dispersion and electrostatic interactions combined with stereochemical effects imposed by the locally strained structural context. They also suggest that the carbon van der Waals (vdW) radii, originally derived for sp3 carbons, should not be used for smaller sp2 carbons attached to electron-withdrawing groups. Using a more adapted carbon vdW radius results in these lp⋅⋅⋅π contacts being no longer of the sub-vdW type. These findings challenge the whole lp⋅⋅⋅π concept that refers to elusive orbital interactions that fail to explain short interatomic contact distances.

Nucleobase carbonyl groups are poor Mg2+ inner-sphere binders but excellent monovalent ion binders-a critical PDB survey.

Precise knowledge of Mg2+ inner-sphere binding site properties is vital for understanding the structure and function of nucleic acid systems. Unfortunately, the PDB, which represents the main source of Mg2+ binding sites, contains a substantial number of assignment issues that blur our understanding of the functions of these ions. Here, following a previous study devoted to Mg2+ binding to nucleobase nitrogens, we surveyed nucleic acid X-ray structures from the PDB with resolutions ≤2.9 Å to classify the Mg2+ inner-sphere binding patterns to nucleotide carbonyl, ribose hydroxyl, cyclic ether, and phosphodiester oxygen atoms. From this classification, we derived a set of "prior-knowledge" nucleobase Mg2+ binding sites. We report that crystallographic examples of trustworthy nucleobase Mg2+ binding sites are fewer than expected since many of those are associated with misidentified Na+ or K+ We also emphasize that binding of Na+ and K+ to nucleic acids is much more frequent than anticipated. Overall, we provide evidence derived from X-ray structures that nucleobases are poor inner-sphere binders for Mg2+ but good binders for monovalent ions. Based on strict stereochemical criteria, we propose an extended set of guidelines designed to help in the assignment and validation of ions directly contacting nucleobase and ribose atoms. These guidelines should help in the interpretation of X-ray and cryo-EM solvent density maps. When borderline Mg2+ stereochemistry is observed, alternative placement of Na+, K+, or Ca2+ must be considered. We also critically examine the use of lanthanides (Yb3+, Tb3+) as Mg2+ substitutes in crystallography experiments.

Identification of receptors for UNCG and GNRA Z-turns and their occurrence in rRNA.

In contrast to GNRA tetraloop receptors that are common in RNA, receptors for the more thermostable UNCG loops have remained elusive for almost three decades. An analysis of all RNA structures with resolution ≤3.0 Å from the PDB allowed us to identify three previously unnoticed receptors for UNCG and GNRA tetraloops that adopt a common UNCG fold, named 'Z-turn' in agreement with our previously published nomenclature. These receptors recognize the solvent accessible second Z-turn nucleotide in different but specific ways. Two receptors participating in a complex network of tertiary interactions are associated with the rRNA UUCG and GAAA Z-turns capping helices H62 and H35a in rRNA large subunits. Structural comparison of fully assembled ribosomes and comparative sequence analysis of >6500 rRNA sequences helped us recognize that these motifs are almost universally conserved in rRNA, where they may contribute to organize the large subunit around the subdomain-IV four-way junction. The third UCCG receptor was identified in a rRNA/protein construct crystallized at acidic pH. These three non-redundant Z-turn receptors are relevant for our understanding of the assembly of rRNA and other long-non-coding RNAs, as well as for the design of novel folding motifs for synthetic biology.

Mg2+ ions: do they bind to nucleobase nitrogens?

Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.

Revisiting GNRA and UNCG folds: U-turns versus Z-turns in RNA hairpin loops.

When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its "natural" fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen-π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for "GNRA-type" folds) and a newly described Z-turn (for "UNCG-type" folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict.

'Z-DNA like' fragments in RNA: a recurring structural motif with implications for folding, RNA/protein recognition and immune response.

Since the work of Alexander Rich, who solved the first Z-DNA crystal structure, we have known that d(CpG) steps can adopt a particular structure that leads to forming left-handed helices. However, it is still largely unrecognized that other sequences can adopt 'left-handed' conformations in DNA and RNA, in double as well as single stranded contexts. These 'Z-like' steps involve the coexistence of several rare structural features: a C2'-endo puckering, a syn nucleotide and a lone pair-π stacking between a ribose O4' atom and a nucleobase. This particular arrangement induces a conformational stress in the RNA backbone, which limits the occurrence of Z-like steps to ≈0.1% of all dinucleotide steps in the PDB. Here, we report over 600 instances of Z-like steps, which are located within r(UNCG) tetraloops but also in small and large RNAs including riboswitches, ribozymes and ribosomes. Given their complexity, Z-like steps are probably associated with slow folding kinetics and once formed could lock a fold through the formation of unique long-range contacts. Proteins involved in immunologic response also specifically recognize/induce these peculiar folds. Thus, characterizing the conformational features of these motifs could be a key to understanding the immune response at a structural level.

Sodium and Potassium Interactions with Nucleic Acids.

Metal ions are essential cofactors for the structure and functions of nucleic acids. Yet, the early discovery in the 70s of the crucial role of Mg(2+) in stabilizing tRNA structures has occulted for a long time the importance of monovalent cations. Renewed interest in these ions was brought in the late 90s by the discovery of specific potassium metal ions in the core of a group I intron. Their importance in nucleic acid folding and catalytic activity is now well established. However, detection of K(+) and Na(+) ions is notoriously problematic and the question about their specificity is recurrent. Here we review the different methods that can be used to detect K(+) and Na(+) ions in nucleic acid structures such as X-ray crystallography, nuclear magnetic resonance or molecular dynamics simulations. We also discuss specific versus non-specific binding to different structures through various examples.

Anions in Nucleic Acid Crystallography.

Nucleic acid crystallization buffers contain a large variety of chemicals fitting specific needs. Among them, anions are often solely considered for pH-regulating purposes and as cationic co-salts while their ability to directly bind to nucleic acid structures is rarely taken into account. Here we review current knowledge related to the use of anions in crystallization buffers along with data on their biological prevalence. Chloride ions are frequently identified in crystal structures but display low cytosolic concentrations. Hence, they are thought to be distant from nucleic acid structures in the cell. Sulfate ions are also frequently identified in crystal structures but their localization in the cell remains elusive. Nevertheless, the characterization of the binding properties of these ions is essential for better interpreting the solvent structure in crystals and consequently, avoiding mislabeling of electron densities. Furthermore, understanding the binding properties of these anions should help to get clues related to their potential effects in crowded cellular environments.

A comprehensive classification and nomenclature of carboxyl-carboxyl(ate) supramolecular motifs and related catemers: implications for biomolecular systems.

Carboxyl and carboxylate groups form important supramolecular motifs (synthons). Besides carboxyl cyclic dimers, carboxyl and carboxylate groups can associate through a single hydrogen bond. Carboxylic groups can further form polymeric-like catemer chains within crystals. To date, no exhaustive classification of these motifs has been established. In this work, 17 association types were identified (13 carboxyl-carboxyl and 4 carboxyl-carboxylate motifs) by taking into account the syn and anti carboxyl conformers, as well as the syn and anti lone pairs of the O atoms. From these data, a simple rule was derived stating that only eight distinct catemer motifs involving repetitive combinations of syn and anti carboxyl groups can be formed. Examples extracted from the Cambridge Structural Database (CSD) for all identified dimers and catemers are presented, as well as statistical data related to their occurrence and conformational preferences. The inter-carboxyl(ate) and carboxyl(ate)-water hydrogen-bond properties are described, stressing the occurrence of very short (strong) hydrogen bonds. The precise characterization and classification of these supramolecular motifs should be of interest in crystal engineering, pharmaceutical and also biomolecular sciences, where similar motifs occur in the form of pairs of Asp/Glu amino acids or motifs involving ligands bearing carboxyl(ate) groups. Hence, we present data emphasizing how the analysis of hydrogen-containing small molecules of high resolution can help understand structural aspects of larger and more complex biomolecular systems of lower resolution.

Protein capsules assembled via isobutyramide grafts: sequential growth, biofunctionalization, and cellular uptake.

We report the sequential assembly of proteins via the alternating physical adsorption of human serum albumin (HSA) and chemical grafting with isobutyramide (IBAM) or bromoisobutyramide (BrIBAM) groups. This approach, performed on silica template particles, leads to the formation of noncovalent protein films with controlled growth at the nanometer scale. Further, after template removal, hollow protein capsules with tunable wall thicknesses and high mechanical stability are obtained. The use of BrIBAM, compared to IBAM grafts, leads to significantly thicker capsule walls, highlighting the influence of the bromine atoms in the assembly process, which is discussed in terms of a theoretical model of noncovalent interactions. Another feature of the process is the possibility to functionalize the HSA capsules with other biologically active macromolecules, including enzymes, polysaccharides, or DNA plasmids, demonstrating the versatility of this approach. We also report that BrIBAM-HSA and IBAM-HSA capsules display negligible cytotoxicity in vitro with HeLa cells and that their cellular uptake is dependent on the thickness of the capsule walls. These findings support the potential use of these protein capsules in tailored biological applications such as drug delivery.

Metal ion binding to RNA.

RNA crystal structures have provided a wealth of information on localized metal ions that are bound to specific sites, such as the RNA deep groove, the Hoogsteen face of guanine nucleotides and anionic phosphate oxygens. With a number of crystal structures being solved with heavy metal derivatives and other "reporter" ions, sufficient information is available to estimate global similarities and differences in ion binding properties and to begin determining the influence of RNA and ions on each other. Here we will discuss the ions that are observed bound to RNA, their coordination properties, and the roles they play in RNA structural studies. Analysis of the crystallographic data reinforces the fact that ion interactions with nucleic acids are not easily interchanged between similarly charged ions. The physiological relevance of RNA-ion interactions, mainly involving K+ and Mg2+ cations, needs to be analyzed with care as different structures are solved under very diverse ionic conditions. The analysis is complicated by the fact that the assignment is not always accurate, often done under sub-optimal conditions, which further limits the generalization about the types of interactions these ions can establish.

A short guide for molecular dynamics simulations of RNA systems.

As a result of important methodological advances and of the rapid growth of experimental data, the number of molecular dynamics (MD) simulations related to RNA systems has significantly increased. However, such MD simulations are not straightforward and great care has to be exerted during the setup stage in order to choose the appropriate MD package, force fields and ionic conditions. Furthermore, the choice and a correct evaluation of the main characteristics of the starting structure are primordial for the generation of informative and reliable MD trajectories since experimental structures are not void of inaccuracies and errors. The aim of this review is to provide, through numerous examples, practical guidelines for the setup of MD simulations, the choice of ionic conditions and the detection and correction of experimental inaccuracies in order to start MD simulations of nucleic acid systems under the best auspices.

Dinucleotide TpT and its 2'-O-Me analogue possess different backbone conformations and flexibilities but similar stacked geometries.

UV irradiation at 254 nm of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a) and of natural thymidylyl(3'-5')thymidine (1b) generates the same photoproducts (CPD and (6-4)PP; responsible for cell death and skin cancer). The ratios of quantum yields of photoproducts obtained from 1a (determined herein) to that from 1b are in a proportion close to the approximately threefold increase of stacked dinucleotides for 1a compared with those of 1b (from previous circular dichroism results). 1a and 1b however are endowed with different predominant sugar conformations, C3'-endo (1a) and C2'-endo (1b). The present investigation of the stacked conformation of these molecules, by unrestrained state-of-the-art molecular simulation in explicit solvent and salt, resolves this apparent paradox and suggests the following main conclusions. Stacked dinucleotides 1a and 1b adopt the main characteristic features of a single-stranded A and B form, respectively, where the relative positions of the backbone and the bases are very different. Unexpectedly, the geometry of the stacking of two thymine bases, within each dinucleotide, is very similar and is in excellent agreement with photochemical and circular dichroism results. Analyses of molecular dynamics trajectories with conformational adiabatic mapping show that 1a and 1b explore two different regions of conformational space and possess very different flexibilities. Therefore, even though their base stacking is very similar, these molecules possess different geometrical, mechanical, and dynamical properties that may account for the discrepancy observed between increased stacking and increased photoproduct formations. The computed average stacked conformations of 1a and 1b are well-defined and could serve as starting models to investigate photochemical reactions with quantum dynamics simulations.

Nucleic acid solvation: from outside to insight.

Nucleic acids are polyanionic molecules that were historically considered to be solely surrounded by a shell of water molecules and a neutralizing cloud of monovalent and divalent cations. In this respect, recent experimental and theoretical reports demonstrate that water molecules within complex nucleic acid structures can display very long residency times, and assist drug binding and catalytic reactions. Finally, anions can also bind to these polyanionic systems. Many of these recent insights are provided by state-of-the-art molecular dynamics simulations of nucleic acid systems, which will be described together with relevant methodological issues.