Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.     

Amino acid modifications on tRNA

The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation. Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa-tRNA synthetases. However, in the case of four amino acids (Gln, Asn, Cys and Sec), aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life. The process begins with the charging of noncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNA(Cys) formation or by synthetases with relaxed specificity, such as the non-discriminating glutamyl-tRNA, non-discriminating aspartyl-tRNA and seryl-tRNA synthetases. The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA, which is catalyzed by a group of tRNA-dependent modifying enzymes, such as tRNA-dependent amidotransferases, Sep-tRNA:Cys-tRNA synthase, O-phosphoseryl-tRNA kinase and Sep-tRNA:Sec-tRNA synthase. The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.     

The occurrence order and cross-talk of different tRNA modifications

Chemical modifications expand the composition of RNA molecules from four standard nucleosides to over 160 modified nucleosides, which greatly increase the complexity and utility of RNAs. Transfer RNAs(tRNAs) are the most heavily modified cellular RNA molecules and contain the largest variety of modifications. Modification of tRNAs is pivotal for protein synthesis and also precisely regulates the noncanonical functions of tRNAs. Defects in tRNA modifications lead to numerous human diseases. Up to now, more than 100 types of modifications have been found in tRNAs. Intriguingly, some modifications occur widely on all tRNAs, while others only occur on a subgroup of tRNAs or even only a specific tRNA. The modification frequency of each tRNA is approximately 7% to 25%, with 5–20 modification sites present on each tRNA. The occurrence and modulation of tRNA modifications are specifically noticeable as plenty of interplays among different sites and modifications have been discovered. In particular, tRNA modifications are responsive to environmental changes, indicating their dynamic and highly organized nature. In this review, we summarized the known occurrence order, cross-talk, and cooperativity of tRNA modifications.     

The role of modifications in codon discrimination by tRNA(Lys)UUU

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.     

Flexibility of ras lipid modifications studied by 2H solid-state NMR and molecular dynamics simulations

Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.     

Analysis of queuosine and 2-thio tRNA modifications by high throughput sequencing

Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas in mammals the substrate for Q-modification in tRNA is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing from biological RNA samples. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm⁵s²U, mcm⁵s²U, cmnm⁵s²U, and s²C by sequencing in human and E. coli tRNA. We term this method periodate-dependent analysis of queuosine and sulfur modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.     

Biosynthesis and function of tRNA modifications in Archaea

tRNA modifications are important for decoding, translation accuracy, and structural integrity of tRNAs. Archaeal tRNAs contain at least 47 different tRNA modifications, some of them, including archaeosine, agmatidine, and mimG, are specific to the archaeal domain. The biosynthetic pathways for these complex signature modifications have recently been elucidated and are extensively described in this review. Archaeal organisms still lag Escherichia coli and Saccharomyces cerevisiae in terms of genetic characterization and in vivo function of tRNA modifications. However, recent advances in the model Haloferax volcanii, described here, should allow closing this gap soon. Consequently, an update on experimental characterizations of archaeal tRNA modification genes and proteins is given to set the stage for future work in this field.     

The emerging impact of tRNA modifications in the brain and nervous system

A remarkable number of neurodevelopmental disorders have been linked to defects in tRNA modifications. These discoveries place tRNA modifications in the spotlight as critical modulators of gene expression pathways that are required for proper organismal growth and development. Here, we discuss the emerging molecular and cellular functions of the diverse tRNA modifications linked to cognitive and neurological disorders. In particular, we describe how the structure and location of a tRNA modification influences tRNA folding, stability, and function. We then highlight how modifications in tRNA can impact multiple aspects of protein translation that are instrumental for maintaining proper cellular proteostasis. Importantly, we describe how perturbations in tRNA modification lead to a spectrum of deleterious biological outcomes that can disturb neurodevelopment and neurological function. Finally, we summarize the biological themes shared by the different tRNA modifications linked to cognitive disorders and offer insight into the future questions that remain to decipher the role of tRNA modifications. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.     

Diagnosis of minor chromosome modifications by molecular cytogenetics

Chromosomal in situ hybridization with radioactive probes allows the detection of single copy DNA segments of very small size. In two phenotypically normal individuals, classical chromosomal analysis revealed a small partial deletion of chromosome 18 short arm. In situ hybridization of probe D18S3, located at 18p13, showed in both instances a balanced reciprocal translocation: 46,XY,t(5;18) (p153;p112) and 46,XX,t(18;22)(p111;p11), respectively.     

The enzymatic mechanism of carboxypeptidase: A molecular dynamics study

An MD simulation of the system carboxypeptidase A (CPA) with the tetrapeptide Val-Leu-Phe-Phe has been performed in order to learn about the substrate disposition just prior to nucleophilic attack. We have explored the model in which the substrate does not substitute the zinc-coordinated water (the “water” mechanism). The simulations do suggest as feasible that the Zn-OH₂ group performs a nucleophilic attack on the Phe-Phe peptidic bond. We have also investigated the model in which the carbonyl oxygen displaces the zinc-coordinated water. In this case the substrate and Glu-270 orient themselves to allow an anhydride intermediate during the peptidic bond cleavage (the “anhydride” mechanism). Based on the results of the simulations, both “water” and “anhydride” mechanisms are structurally feasible, although the former model seems more probable on chemical grounds. © 1994 John Wiley & Sons, Inc.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.     

Crystal morphology study of N,N'-diacetylchitobiose by molecular dynamics simulation

Bisphenoidal shape of α-N,N′-diacetylchitobiose (α-(GlcNAc)₂) monohydrate crystals was obtained from aqueous solution. Crystal morphology was studied by computer simulation. Theoretical morphologies calculated by classic models (a BFDH model, a surface free-energy method, and an AE model) deviated significantly from that from the experimental crystal habit. Therefore, a solvent effect was considered by introducing an interface layer model based on molecular dynamics simulation in order to bridge this gap. The results of the simulation showed that the calculated habit is much closer to the experiment, meaning that the interface layer model describes the solvent effect very well. This model may also be applied for other oligosaccharide systems to study the relationship among crystal morphology, crystal structure, and solvation effects.     

Structure-activity study of endomorphin-2 analogs with C-terminal modifications by NMR spectroscopy and molecular modeling

Endomorphin-2 (EM-2) is a putative endogenous mu-opioid receptor ligand. To get insight into the important role of C-terminal amide group of EM-2, we investigated herein a series of EM-2 analogs by substitution of the C-terminal amide group with -NHNH(2), -NHCH(3), -N(CH(3))(2), -OCH(3), -OCH(2)CH(3), -OC(CH(3))(3), and -CH(2)-OH. Their binding affinity and bioactivity were determined and compared. Despite similar (analogs 1, 4, and 7) or decreased (analogs 2, 3,5, and 6) mu affinity in binding assays, all analogs showed low guinea pig ileum (GPI) and mouse vas deferens (MVD) potencies compared to their parent peptide. Interestingly, as for analogs 2 and 3 (a single and double N-methylation of C-terminal amide), the potency order with the K(i) (mu) values was 2>3; for the C-terminal esterified analogs 4-6, the potency order with the K(i) (mu) values was 4>5>6. Thus, we concluded that the steric hindrance of C-terminus might play an important role in opioid receptor affinity. We further investigated the conformational properties of these analogs by 1D and 2D (1)H NMR spectroscopy and molecular modeling. Evaluating the ratios of cis- and trans-isomers, aromatic interactions, dihedral angles, and stereoscopic views of the most convergent conformers, we found that modifications at the C-terminal amide group of EM-2 affected these analog conformations markedly, therefore changed the opioid receptor affinity and in vitro bioactivity.