Discovering structural motifs using a structural alphabet: Application to magnesium-binding sites

Background  

For many metalloproteins, sequence motifs characteristic of metal-binding sites have not been found or are so short that they would not be expected to be metal-specific. Striking examples of such metalloproteins are those containing Mg²⁺, one of the most versatile metal cofactors in cellular biochemistry. Even when Mg²⁺-proteins share insufficient sequence homology to identify Mg²⁺-specific sequence motifs, they may still share similarity in the Mg²⁺-binding site structure. However, no structural motifs characteristic of Mg²⁺-binding sites have been reported. Thus, our aims are (i) to develop a general method for discovering structural patterns/motifs characteristic of ligand-binding sites, given the 3D protein structures, and (ii) to apply it to Mg²⁺-proteins sharing <30% sequence identity. Our motif discovery method employs structural alphabet encoding to convert 3D structures to the corresponding 1D structural letter sequences, where the Mg²⁺-structural motifs are identified as recurring structural patterns.     

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg²⁺-μc's). Common features of Mg²⁺-μc's include two paired Mg²⁺ ions that are chelated by a common bridging phosphate group in the form Mg_(a)²⁺–(O1P-P-O2P)–Mg_(b)²⁺. This bridging phosphate is part of a 10-membered chelation ring in the form Mg_(a)²⁺–(OP-P-O5′-C5′-C4′-C3′-O3′-P-OP)–Mg_(a)²⁺. The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg²⁺ ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg²⁺-μc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg²⁺-μc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg²⁺-μc's in other rRNAs including the bacterial 16S rRNA, and the P4–P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg²⁺-μc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.     

Reduced Model Captures Mg2+-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.     

Predicting Ion Binding Properties for RNA Tertiary Structures

Recent experiments pointed to the potential importance of ion correlation for multivalent ions such as Mg²⁺ ions in RNA folding. In this study, we develop an all-atom model to predict the ion electrostatics in RNA folding. The model can treat ion correlation effects explicitly by considering an ensemble of discrete ion distributions. In contrast to the previous coarse-grained models that can treat ion correlation, this new model is based on all-atom nucleic acid structures. Thus, unlike the previous coarse-grained models, this new model allows us to treat complex tertiary structures such as HIV-1 DIS type RNA kissing complexes. Theory-experiment comparisons for a variety of tertiary structures indicate that the model gives improved predictions over the Poisson-Boltzmann theory, which underestimates the Mg²⁺ binding in the competition with Na⁺. Further systematic theory-experiment comparisons for a series of tertiary structures lead to a set of analytical formulas for Mg²⁺/Na⁺ ion-binding to various RNA and DNA structures over a wide range of Mg²⁺ and Na⁺ concentrations.     

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.     

Reduced Model Captures Mg-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

The bulge region of HIV-1 TAR RNA binds metal ions in solution

Binding of Mg²⁺, Ca²⁺ and Co(NH₃)₆³⁺ ions to the HIV-1 TAR RNA in solution was analysed by ¹⁹F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for ¹⁹F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg²⁺ and Ca²⁺ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K_d) of 0.9 ± 0.6 and 2.7 ± 1.7 mM, respectively. Argininamide, inducing largest ¹⁹F chemical shifts changes at FU-23, was used as a reference ligand (K_d = 0.3 ± 0.1 mM). In the Pb²⁺-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg²⁺ and Ca²⁺ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb²⁺-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH₃)₆³⁺ > Mg²⁺ > Ca²⁺] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.     

Salt dependence of nucleic acid hairpin stability

Single-stranded junctions/loops are frequently occurring structural motifs in nucleic acid structures. Due to the polyanionic nature of the nucleic acid backbone, metal ions play a crucial role in the loop stability. Here we use the tightly bound ion theory, which can account for the possible ion correlation and ensemble (fluctuation) effects, to predict the ion-dependence of loop and stem-loop (hairpin) free energies. The predicted loop free energy is a function of the loop length, the loop end-to-end distance, and the ion (Na⁺ and Mg²⁺ in this study) concentrations. Based on the statistical mechanical calculations, we derive a set of empirical formulas for the loop thermodynamic parameters as functions of Na⁺ and Mg²⁺ concentrations. For three specific types of loops, namely, hairpin, bulge, and internal loops, the predicted free energies agree with the experimental data. Further applications of these empirical formulas to RNA and DNA hairpin stability lead to good agreements with the available experimental data. Our results indicate that the ion-dependent loop stability makes significant contribution to the overall ion-dependence of the hairpin stability.     

Microsecond Molecular Dynamics Simulations of Mg2+- and K+- Bound E1 Intermediate States of the Calcium Pump

We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca²⁺-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg²⁺ in the absence of Ca²⁺ has shown that SERCA adopts an E1 structure with transmembrane Ca²⁺-binding sites I and II exposed to the cytosol, stabilized by a single Mg²⁺ bound to a hybrid binding site I′. This Mg²⁺-bound E1 intermediate state, designated E1•Mg²⁺, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca²⁺ by facilitating H⁺/Ca²⁺ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg²⁺ crystal structure, starting in the presence or absence of initially-bound Mg²⁺. Both simulations were performed for 1 µs in a solution containing 100 mM K⁺ and 5 mM Mg²⁺ in the absence of Ca²⁺, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg²⁺, SERCA site I′ maintained Mg²⁺ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg²⁺, two K⁺ ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg²⁺ and E•2K⁺ intermediate states of SERCA are populated in solution in the absence of Ca²⁺, with the more open 2K⁺-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K⁺ state acts as a functional intermediate that facilitates the E2 to E1•2Ca²⁺ transition through two mechanisms: by pre-organizing transport sites for Ca²⁺ binding, and by partially opening the cytosolic headpiece prior to Ca²⁺ activation of nucleotide binding.     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Effects of Metal-Binding Properties of Human Kv Channel-Interacting Proteins on their Molecular Structure and Binding with Kv4.2 Channel

The goal of the present study is to explore whether Ca²⁺ and Mg²⁺-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca²⁺-binding sites, respectively, and only one type of Mg²⁺-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca²⁺, but the binding affinity for Mg²⁺ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg²⁺ and Ca²⁺. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg²⁺ and Ca²⁺. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg²⁺- and Ca²⁺-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.     

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg²⁺, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg²⁺ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg²⁺-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg²⁺, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg²⁺ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg²⁺-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg²⁺-ion interactions contribute to RNA structural properties and stability.     

Localization and functional characterization of metal-binding sites in phytochelatin synthases

Metal-binding domains consisting of short, contiguous stretches of amino acids are found in many proteins mediating the transport, buffering, trafficking or detoxification of metal ions. Phytochelatin synthases are metal-activated enzymes that function in the detoxification of Cd²⁺ and other toxic metal and metalloid ions. In order to localize Cd²⁺-binding sites, peptide libraries of two diverse phytochelatin synthases were synthesized and incubated with ¹⁰⁹Cd²⁺. Distinct binding sites and binding motifs could be localized based on the patterns of Cd²⁺-binding. The number of binding sites was consistent with previous findings for recombinant protein. Positions of binding sites appeared to be conserved even among diverse phytochelatin synthases. Mutant peptide analysis was used to assess the contribution of exemplary amino acids to binding. Several binding motifs contain cysteines or glutamates. For cysteines a strong correlation was found between binding activity and degree of conservation among known phytochelatin synthases. These findings indicate the suitability of peptide scanning for the identification of metal-binding sites. The functional role of several cysteines was investigated by expression of hemagglutinin-tagged phytochelatin synthases in phytochelatin synthase-deficient, Cd²⁺-hypersensitive Schizosaccharomyces pombe cells. The data are consistent with a model suggesting functionally essential metal-binding activation sites in the N-terminal catalytic part of phytochelatin synthases and additional binding sites at the C-terminus not essential for activity.Abbreviations EMM Edinburgh's minimal medium - GSH glutathione - HA hemagglutinin - PC phytochelatin - PCS phytochelatin synthase     

Structural insights of HutP-mediated regulation of transcription of the hut operon in Bacillus subtilis

Regulating gene expression directly at the mRNA level represents a novel approach to control cellular processes in all organisms. In this respect, an RNA-binding protein plays a key role by targeting the mRNA to regulate the expression by attenuation or an anti-termination mechanism only in the presence of their cognate ligands. Although many proteins are known to use these mechanisms to regulate the gene expression, no structural insights have been revealed to date to explain how these proteins trigger the conformation for the recognition of RNA. This review describes the activated conformation of HutP, brought by the coordination of L-histidine and Mg²⁺ ions, based on our recently solved crystal structures [uncomplexed HutP, HutP–Mg²⁺, HutP–L-histidine, HutP–Mg²⁺–L-histidine, HutP–Mg²⁺–L-histidine-RNA]. Once the HutP is activated, the protein binds specifically to bases within the terminator region, without undergoing further structural rearrangement. Also, a high resolution (1.48 Å) crystal structure of the quaternary complex containing the three GAG motifs is presented. This analysis clearly demonstrates that the first base in the UAG motifs is not important for the function and is consistent with our previous observations.     

Magnetic isotope and magnetic field effects on the DNA synthesis

Magnetic isotope and magnetic field effects on the rate of DNA synthesis catalysed by polymerases β with isotopic ions ²⁴Mg²⁺, ²⁵Mg²⁺ and ²⁶Mg²⁺ in the catalytic sites were detected. No difference in enzymatic activity was found between polymerases β carrying ²⁴Mg²⁺ and ²⁶Mg²⁺ ions with spinless, non-magnetic nuclei ²⁴Mg and ²⁶Mg. However, ²⁵Mg²⁺ ions with magnetic nucleus ²⁵Mg were shown to suppress enzymatic activity by two to three times with respect to the enzymatic activity of polymerases β with ²⁴Mg²⁺ and ²⁶Mg²⁺ ions. Such an isotopic dependence directly indicates that in the DNA synthesis magnetic mass-independent isotope effect functions. Similar effect is exhibited by polymerases β with Zn²⁺ ions carrying magnetic ⁶⁷Zn and non-magnetic ⁶⁴Zn nuclei, respectively. A new, ion–radical mechanism of the DNA synthesis is suggested to explain these effects. Magnetic field dependence of the magnesium-catalysed DNA synthesis is in a perfect agreement with the proposed ion–radical mechanism. It is pointed out that the magnetic isotope and magnetic field effects may be used for medicinal purposes (trans-cranial magnetic treatment of cognitive deceases, cell proliferation, control of the cancer cells, etc).     

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg²⁺-ions. Furthermore, a role for Mg²⁺-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg²⁺, but becomes partially pre-organized for ligand binding in the presence of Mg²⁺. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg²⁺ is more pronounced.     

Interactions of noncanonical motifs with hnRNP A2 promote activity-dependent RNA transport in neurons

A key determinant of neuronal functionality and plasticity is the targeted delivery of select ribonucleic acids (RNAs) to synaptodendritic sites of protein synthesis. In this paper, we ask how dendritic RNA transport can be regulated in a manner that is informed by the cell’s activity status. We describe a molecular mechanism in which inducible interactions of noncanonical RNA motif structures with targeting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 form the basis for activity-dependent dendritic RNA targeting. High-affinity interactions between hnRNP A2 and conditional GA-type RNA targeting motifs are critically dependent on elevated Ca²⁺ levels in a narrow concentration range. Dendritic transport of messenger RNAs that carry such GA motifs is inducible by influx of Ca²⁺ through voltage-dependent calcium channels upon β-adrenergic receptor activation. The combined data establish a functional correspondence between Ca²⁺-dependent RNA–protein interactions and activity-inducible RNA transport in dendrites. They also indicate a role of genomic retroposition in the phylogenetic development of RNA targeting competence.     

Docking of calcium ions in proteins with flexible side chains and deformable backbones

A method of docking Ca²⁺ ions in proteins with flexible side chains and deformable backbones is proposed. The energy was calculated with the AMBER force field, implicit solvent, and solvent exposure-dependent and distance-dependent dielectric function. Starting structures were generated with Ca²⁺ coordinates and side-chain torsions sampled in 1000 Å³ cubes centered at the experimental Ca²⁺ positions. The energy was Monte Carlo-minimized. The method was tested on fourteen Ca²⁺-binding sites. For twelve Ca²⁺-binding sites the root mean square (RMS) deviation of the apparent global minimum from the experimental structure was below 1.3 and 1.7 Å for Ca²⁺ ions and side-chain heavy atoms, respectively. Energies of multiple local minima correlate with the RMS deviations from the X-ray structures. Two Ca²⁺-binding sites at the surface of proteinase K were not predicted, because of underestimation of Ca²⁺ hydration energy by the implicit-solvent method.