Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA^Phe was studied. Removal of domain 8 of the β subunit resulted in drastic reduction of the dissociation constant of the FRS·tRNA^Phe complex. Neither the removal of arginine 2 of the β subunit, which makes the only major contact between domains β1–5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1–5 of the β subunit may not be essential for efficient aminoacylation of tRNA^Phe by the FRS and rather be involved in other functions.     

The Seryl-tRNA synthetase/tRNA acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA^Ser. The Seryl-tRNA-synthetase interacts with the tRNA^Ser acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein–RNA complex. We solved the high resolution crystal structures of two tRNA^Ser acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.     

Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress

Background

Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis.

Structural changes of tRNA and 5S rRNA induced with magnesium and visualized with synchrotron mediated hydroxyl radical cleavage

The structure of native yeast tRNA^Phe and wheat germ ribosomal 5S RNA induced by different magnesium ion concentrations was studied in solution with a synchrotron mediated hydroxyl radical RNA cleavage reaction. We showed that very small amounts of Mg⁺² can induce significant changes in the hydroxyl radical cleavage pattern of tRNA^Phe. It also turned out that a reactivity of tRNA^Phe towards OH coincides with the strong metal binding sites. Because of the Mg ions are heavily hydrated one can suggest the strong correlation of the observed nucleosides reactivity in vicinity of Mg²⁺ binding sites with availability of water molecules as a source of hydroxyl radical. On the other hand the structure of wheat germ 5S rRNA is less sensitive to the hydroxyl radical reaction than tRNA^Phe although some changes are visible at 4 mM Mg ions. It is probably due to the lack of strong Mg⁺² binding sites in that molecule. The reactivity of nucleotides in loops C and D of 5S rRNA is not effected, what suggests their flexibility or involvement in higher order structure formation. There is different effect of magnesium on tRNA and 5S rRNA folding. We found that nucleotides forming strong binding sites for magnesium are very sensitive to X-ray generated hydroxyl radical and can be mapped with OH. The results show, that guanine nucleotides are preferentially hydrated. X-ray footprinting mediated hydroxyl radical RNA cleavage is a very powerful method and has been applied to studies of stable RNAs for the first time.     

Crystal structure of a human tRNA(Gly) microhelix at 1.2A resolution

The tRNA^Gly/glycyl-tRNA synthetase (GlyRS) system belongs to the so-called ‘class II aminoacyl-tRNA synthetase system’ in which tRNA identity elements are assured by rather few and simple determinants mostly located in the tRNA acceptor stem. Regarding evolutionary aspects, the tRNA^Gly/GlyRS system is a special case. There exist two different types of GlyRS, namely an archaebacterial/human type and a eubacterial type reflecting an evolutionary divergence within this system.Here we report the crystal structure of a human tRNA^Gly acceptor stem microhelix at 1.2 Å resolution. The local geometric parameters of the microhelix and the water network surrounding the RNA are presented. The structure complements the previously published Escherichia coli tRNA^Gly aminoacyl stem structure.     

Comparative X-ray structure analysis of human and Escherichia coli tRNA(Gly) acceptor stem microhelices

tRNA identity elements assure the correct aminoacylation of tRNAs by the aminoacyl-tRNA synthetases with the cognate amino acid. The tRNA^Gly/glycyl-tRNA sythetase system is member of the so-called ‘class II system’ in which the tRNA determinants consist of rather simple elements. These are mostly located in the tRNA acceptor stem and in the glycine case additionally the discriminator base at position 73 is required. Within the glycine-tRNA synthetases, the archaebacterial/human and the eubacterial sytems differ with respect to their protein structures and the required tRNA identity elements, suggesting a unique evolutionary divergence.In this study, we present a comparison between the crystal structures of the eubacterial Escherichia coli and the human tRNA^Gly acceptor stem microhelices and their surrounding hydration patterns.     

Crystallization and preliminary X-ray diffraction analysis of <Emphasis Type="Italic">E. coli</Emphasis> arginyl-tRNA synthetase in complex form with a tRNA<Superscript>Arg</Superscript>

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA(Arg) (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 A and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 A, c = 463.2 A, and alpha = beta = 90 degrees , gamma = 120 degrees , being different from a = b = 110.8 A, c = 377.8 A for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.     

The influence of beta-arrestin2 on cannabinoid CB1 receptor coupling to G-proteins and subcellular localization and relative levels of beta-arrestin1 and 2 in mouse brain

Abstract

Context: Beta-arrestins are known to couple to some G-protein-coupled receptors (GPCRs) to regulate receptor internalization, G-protein coupling and signal transduction, but have not been investigated for most receptors, and for very few receptors in vivo. Previous studies have shown that beta-arrestin2 deletion enhances the efficacy of specific cannabinoid agonists. Objective: The present study hypothesized that brain cannabinoid CB₁ receptors are regulated by beta-arrestin2. Methods: Beta-arrestin2+/+ and ?/? mice were used. Western blotting was used to determine the relative levels of each beta-arrestin subtype in mouse brain. Receptor binding was measured to determine whether deletion of beta-arrestin2 influences agonist binding to brain CB₁ receptors, or the subcellular localization of CB₁ in brain membranes subjected to differential centrifugation. A variety of cannabinoid agonists from different chemical classes were investigated for their ability to activate G-proteins in the presence and absence of beta-arrestin2 in cerebellum, hippocampus and cortex. Results: No differences were found in the density of beta-arrestin1 or cannabinoid CB₁ receptors in several brains of beta-arrestin2+/+ versus ?/? mice. Differences between genotypes were found in the proportion of high- and low-affinity agonist binding sites in brain areas that naturally express higher levels of beta-arrestin2. Cortex from beta-arrestin2?/? mice contained less CB₁ in the P1 fraction and more CB₁ in the P2 fraction compared to beta-arrestin2+/+. Of the agonists assayed for activity, only Δ⁹-tetrahydrocannabinol (THC) exhibited a difference between genotypes, in that it was less efficacious in beta-arrestin2?/? than +/+ mouse membranes. Conclusion: Beta-arrestin2 regulates cannabinoid CB₁ receptors in brain.     

The crystal structure of a Thermus thermophilus tRNA acceptor stem microhelix at 1.6 Å resolution

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem.The tRNA^Gly/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA^Gly acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA^Gly aminoacyl stem from Thermus thermophilus at 1.6 Å resolution and provide insight into the RNA geometry and hydration.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Structural basis of the water-assisted asparagine recognition by asparaginyl-tRNA synthetase

Asparaginyl-tRNA synthetase (AsnRS) is a member of the class-II aminoacyl-tRNA synthetases, and is responsible for catalyzing the specific aminoacylation of tRNA(Asn) with asparagine. Here, the crystal structure of AsnRS from Pyrococcus horikoshii, complexed with asparaginyl-adenylate (Asn-AMP), was determined at 1.45 A resolution, and those of free AsnRS and AsnRS complexed with an Asn-AMP analog (Asn-SA) were solved at 1.98 and 1.80 A resolutions, respectively. All of the crystal structures have many solvent molecules, which form a network of hydrogen-bonding interactions that surrounds the entire AsnRS molecule. In the AsnRS/Asn-AMP complex (or the AsnRS/Asn-SA), one side of the bound Asn-AMP (or Asn-SA) is completely covered by the solvent molecules, which complement the binding site. In particular, two of these water molecules were found to interact directly with the asparagine amide and carbonyl groups, respectively, and to contribute to the formation of a pocket highly complementary to the asparagine side-chain. Thus, these two water molecules appear to play a key role in the strict recognition of asparagine and the discrimination against aspartic acid by the AsnRS. This water-assisted asparagine recognition by the AsnRS strikingly contrasts with the fact that the aspartic acid recognition by the closely related aspartyl-tRNA synthetase is achieved exclusively through extensive interactions with protein amino acid residues. Furthermore, based on a docking model of AsnRS and tRNA, a single arginine residue (Arg83) in the AsnRS was postulated to be involved in the recognition of the third position of the tRNA(Asn) anticodon (U36). We performed a mutational analysis of this particular arginine residue, and confirmed its significance in the tRNA recognition.     

Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride     

Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery

The relative water content (RWC), free proline levels and the activities of enzymes involved in proline metabolism were studied in drought tolerant (Ca/H 680) and drought sensitive (Ca/H 148) genotypes of cotton (Gossypium hirsutum L.) during induction of water stress and posterior recovery. Water stress caused a significant increase in proline levels and P5CS activity in leaves of both tolerant and sensitive genotypes, whereas the activity of P5CR increased minimally and the activity of OAT remains unchanged. The activity of PDH decreased under drought stress in both the genotypes. The leaf of tolerant genotype maintained higher RWC, photosynthetic activity and proline levels, as well as higher P5CS and P5CR activities under water stress than that of drought sensitive genotype. The drought induced proline levels and activities of P5CS and P5CR declined and tend to be equal to their respective controls, during recovery, whereas the PDH activity tends to increase. These results indicate that induction of proline levels by up regulation of P5CS and down regulation of PDH may be involved in the development of drought tolerance in cotton.     

The Ca2+-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes,consistent with a 2 Ca2+/nH+ exchange mechanism

The Ca²⁺-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca²⁺ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pH_cyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca²⁺ to 2.5 μM and monitoring active Ca²⁺ extrusion into a Ca²⁺-free medium. The pump was shown to perturb pH_cyt, to not respond to changes in membrane potential and to respond to imposed changes in pH_cyt in a manner consistent with the Ca²⁺ pump acting as a 2 Ca²⁺/nH⁺ exchanger. (i) Raising the external pH (pH_ext) from 7.40 to 7.60 lowers the V_max of the pump in basal condition (V_max,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pH_ext to 7.13 raised V_max,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG⁺) medium, the pump operation against high [Ca²⁺]_cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pH_cyt down to 6.23 reduces the V_max,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na⁺ raises the V_max,1 (basal state withK _m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (V_max,2=280±28 μM/min;K _m,2=502±36 nM). (vi) Transient elevation of pH_cyt by NH₄Cl at high [Ca²⁺]_cyt activates the pump eightfold (V_max,2≥671±350 μM/min). The large activation by alkaline pH_cyt at high [Ca²⁺]_cyt can be explained by Ca²⁺-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca²⁺ affinity of calmodulin at high pH.     

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV''s NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.     

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.