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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Valyl-tRNA Synthetases

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Valyl-tRNA synthetase (ValRS), a large monomeric enzyme in a free state, forms a class-Ia subfamily, which characteristically have an α-helix bundle domain near the C-terminus to recognize the tRNA anticodon. The previous mutagenesis studies on tRNAVal identified A35, C36, nucleotide at position 20, and G45 as identity elements, with the latter two being minor determinants. The 2.9-Å resolution crystal structure of Thermus thermophilus ValRS in a complex with tRNAVal and valyl-adenylate analogue corroborates the biochemical work. On the other hand, the C-terminal coiled-coil domain of ValRS interacts electrostatically with adenosine at position 20 and hydrophobically with the G19•C56 tertiary base pair. The interactions between ValRS and the variable pocket of tRNAVal induce a more open conformation of the TΨC-loop/D-loop core, which may account for the previous results by 19F and imino proton NMR of tRNAVal. ValRS strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent “double sieve” mechanism. The ternary complex structure, representing the post-transfer editing state, substantiates the first, size-based sieve that precludes L-isoleucine, and the second, hydrophilicity-based sieve that deacylates threonyl product.

An Overview of ValRS

The gene for valyl-tRNA synthetase (ValRS) (valS) from Escherichia coli was first cloned in 1984.1 In 1987, the yeast ValRS gene (VAS1) was sequenced, and it was reported that the encoded polypeptide shows high homology with IleRS.2 The VAS1 gene was shown to encode both mitochondrial and cytoplasmic ValRSs.3 Further phylogenetic analyses suggested that, in eukaryotic cells, ValRS gene may have been transferred from the mitochondrial genome to the nuclear genome.4 Recent genome analyses also revealed that genes for valyl- and threonyl-tRNA synthetase from Arabidopsis thaliana encode the cytosolic and mitochondrial forms of the enzymes by alternative use of two in-frame initiation codons.5 In mammalian cells, ValRS forms a high Mr complex with the four subunits of elongation factor EF-1H, where EF-1 α regulates the activity of ValRS.6 This finding raises a possibility of functional linkage between aminoacylation and EF-1 α •tRNAVal formation, known as “tRNA channeling”.

ValRS is classified into the class-Ia subgroup together with IleRS, LeuRS, MetRS, CysRS, and ArgRS.7,8 As compared with the other class I subgroups, class Ia synthetases are characterized by two structural domains;9 a helical insertion into the N-terminal half of the canonical Rossmann fold domain, and an α-helix bundle domain, near the C-terminus, involved in recognition of the tRNA anticodon. On the other hand, there are structural features common to class Ia and Ib subgroups such as the connective polypeptide (CP) domain, intervening into the Rossmann fold domain, which has a four-stranded antiparallel β;-sheet core, and the stem-contact-fold (SC-fold) domain with a β.α-α-β. α topology follows the Rossmann fold domain.9 Especially, ValRS, IleRS, and LeuRS are evolutionarily related: they have a large CP domain involved in the proof-reading/editing activity for discrimination of the near-cognate amino acids,10-12 as described below.

tRNA Recognition

tRNAVal Identity

To identify the identity elements of tRNAVal, the Horowitz group analyzed the interactions between Escherichia coli tRNAVal and ValRS by enzymatic footprinting with nuclease S1 and ribonuclease V1,13 and by19 F NMR of 5-fluorouracil-substituted tRNAVal.14,15 The enzymatic footprinting study revealed that ValRS specifically protects the anticodon loop, the 3' side of the stacked TΨC-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases.13 We further analyzed the detailed molecular interactions between tRNAVal and ValRS from E. coli by chemical footprinting with N-nitroso-N-ethylurea, as shown in Figure 1A (Niimi T, Nureki O, Yokoyama S., manuscript in preparation). The anticodon loop, the middle of the D stem, the 3' side of the anticodon stem, and the 3' side of the middle of the acceptor stem were protected by ValRS from the attack of the alkylating reagent (fig.1A). On the other hand, in the enzymatic footprinting, the nuclease susceptibility at the ends of the anticodon- and TΨC-stem helix was increased.13 The chemical footprinting also revealed that the two phosphodiester bonds in the TΨC-loop are more susceptible to the alkylation upon binding to ValRS (fig. 1A). These increased reactivities at the anticodon stem and the TΨC-arm indicate enzyme-induced conformational changes in the tRNA.

Figure 1. A) Phosphate-mapping of E.

Figure 1

A) Phosphate-mapping of E. coli tRNAVal in a complex with ValRS by N-nitroso-N-ethylurea. The solid arrows represent protection from alkylation upon the complex formation. The open arrows represent the phosphates that are more susceptible to alkylation (more...)

On the basis of the footprinting analysis, aminoacylation kinetics of mutant tRNAVal transcripts were carried out.13 The most important synthetase recognition elements are A35 and C36, which were initially reported by the in vivo experiment.16 Nucleotides at position 20, in the variable pocket, and at position 45, in the tRNA central core, are minor identity elements, which was demonstrated by identity switch experiments with E. coli tRNAPhe. Adenosine, cytidine, and uridine at the discriminator position (#73) are readily recognized by ValRS, while G73 acts as a negative determinant.13 The Shimizu group reported that trnasplantation of G3•C70 and U4•A69 base pairs into tRNAAla with UAC anticodon converts the tRNAAla transcript to an efficient substrate of ValRS, suggesting that the two base pairs in the acceptor helix are also minor identity determinants.17

NMR Analyses

Spectrum changes in 19F NMR of 5-fluorouracil-substituted tRNAVal with and without ValRS by the Horowitz group revealed not only the recognition sites by ValRS but also the conformational changes of tRNAVal upon binding with ValRS (fig. 1B).14,15 The most notable changes are the loss of intensity of FU34, FU7, and FU67 as the concentration of ValRS increases, with FU34, at the wobble position of the anticodon, being affected most (fig. 1B). The loss of intensity at these resonances may be ascribed to a gradual peak broadening caused by ValRS binding. Higher concentrations of ValRS produce broadening and downfield shifts of resonances FU12, FU4 and/or FU8 (fig. 1B). ValRS also causes a splitting of resonances FU55 and FU64 in the TΨC-loop and stem of tRNAVal (fig. 1B). Especially, the downfield shift of resonance FU55 upon binding to ValRS may indicate a move of FU55 to a less hydrophobic, more exposed, environment. The 19F NMR result implies conformational changes involving the TΨC-loop and stem of (FUra)tRNAVal on ValRS binding, possibly a (partial) disruption of TΨC-loop/D-loop interactions.

Our imino proton NMR analysis of E. coli tRNAVal showed similar but more detailed conformational changes in tRNAVal upon binding to ValRS (fig. 1B) (Niimi T, Nureki O, Yokoyama S., manuscript in preparation). Disappears of imino proton signal were observed at base pairs of G5•C68 in the acceptor stem, C11•G24 in the D-stem, and C28•G42 and U29•A41 in the anticodon stem (fig. 1B), which suggests significant distortion of these base pairs by ValRS interaction. The broadening of signals of tertiary base pairs of G15•C48, G18•Ψ56, and G19•C56 as well as the chemical-shift changes in tertiary base pairs of s4U8•A14 and G22•m7G46 (fig. 1B) imply that conformational changes involving the tRNA core are induced by the recognition by ValRS. The signal broadening of the stem base pairs of G10•C25, U12•A23, and C30•G40 may result from the recognition of the nearby identity elements by ValRS (fig. 1B).

Crystal Structure of ValRS• tRNAVal Complex

The crystal structure of Thermus thermophilus ValRS was determined in a complex with tRNAVal(CAC) and a Val-AMP analog (Val-AMS) at 2.9 Å resolution,17 and shows architecture conserved in the class-Ia aaRSs for specific tRNA recognition18 (fig. 2A): the stem-contact-fold (SC-fold) domain (colored in red) and the following α-helix bundle domain (colored in violet) are appended to the Rossmann-fold domain.9 In addition, the α-helix bundle domain connects with the following “anticodon-stem-binding” junction domain (referred to hereafter as “AS-binding domain”, colored in aquamarine) and the C-terminal coiled-coil domain (colored in lime green) in ValRS (fig.2A). In the T. thermophilus ValRS•tRNAVal•Val-AMS complex structure, the L-shaped tRNAVal molecule is clamped between the SC-fold domain and the AS-binding and coiled-coil domains on the inner and outer sides, respectively (fig. 2B). The ValRS•tRNAVal complex structure clearly accounts for the aforementioned results of footprinting, mutant kinetics, and NMR analyses. Intriguingly, monomeric ValRS forms a homo dimer, involving the C-terminal coiled-coil domain, to form a 2:2 complex with tRNA, which was also detected by the previous neutron small angle scattering experiment.19

Figure 2. A) Crystal structure of ValRS•tRNAVal complex.

Figure 2

A) Crystal structure of ValRS•tRNAVal complex. The domains are colored according to the following rules: Rossmann-fold (aminoacylation) domain, green; CP core, white; CP1 (editing) domain, cyan; CP2 domain, orange; CP3 domain, yellow; SC-fold (more...)

Interaction of the tRNA D-Stem with the SC-Fold Domain of ValRS

The SC-fold domain of ValRS forms a positively charged patch, which contacts the phosphate backbone of the D-stem of tRNAVal (fig. 2B).18 This contact between the ValRS SC-fold domain and the minor groove side of the tRNAVal D-stem is consistent with the results of the footprinting analysis of E. coli tRNAVal and ValRS (fig. 1A)14,15 (Niimi T, Nureki O, Yokoyama S., manuscript in preparation). The two NηH2 groups of Arg570 hydrogen bond with the O2 atoms of C11 and C25, respectively. The aromatic NH group of Trp571 forms weak hydrogen bonds with O2 and O2' atom of U12. These base-specific recognitions cause distortion of the planarity of the base pairs. The NηH2 group of Arg566 hydrogen bonds with the phosphate group of C13. These recognitions and conformational changes in tRNAVal account for the previous results of chemical footprinting analysis (fig. 1A) (Niimi T, Nureki O, Yokoyama S., manuscript in preparation), and of the 19F and imino proton NMR analyses (fig. 1B).14,15

Recognition of the tRNAVal Anticodon Loop by the α-Helix Bundle Domain of ValRS

The anticodon loop of tRNAVal is extensively deformed, unwound, and bound to the bottom of the α-helix bundle domain on the minor groove side (fig. 2).18 In the ValRS•tRNAVal•Val-AMS complex structure, the intramolecular stacking interactions within the tRNA anticodon loop are disrupted, and the anticodon loop is unwound toward the D-loop side (fig.3A). In the extensively deformed anticodon loop of tRNAVal, C32, U33, and C34 are not well ordered. The first letter of the anticodon (C34) is exposed to the solvent, and is not recognized by ValRS (fig. 3A), which is consistent with the previous in vivo and in vitro mutational results that C34 of tRNAVal is not the identity element.10,13,16 In contrast, A35 and C36 are the major identity elements of tRNAVal.10,13-16 In the ValRS•tRNAVal•Val-AMS complex structure, A35 and C36 form a base-stacking interaction, and fit into a pocket formed by the first and third α-helices of the four-stranded α-helix bundle domain (fig. 3). This stacking interaction between A35 and C36 allows ValRS to recognize the successive AC sequence on the anticodon loop. Most natural tRNA species have neither A34-C35 nor A36-C37, because A34 is always modified to inosine, and position 37 is generally occupied by a purine nucleotide.20,21 Therefore, the recognition of the successive AC sequence around the tRNA anticodon may prevent the misrecognition of the non-cognate tRNAs by ValRS. The A35 base forms van der Waals interactions with the Phe588 and Leu650 side chains (fig. 3). The 6-NH2 group of A35 hydrogen bonds with the α-CO group of Cys646 and the CδOO- group of Glu651. The N3 atom of C36 hydrogen bonds with the NζH2 group of Lys581. The NηH2 group of Arg587 and the NδH2 group of Asn584 hydrogen bond with the 2'-OH groups of A35 and C36, respectively. Glu651 and Lys581 are completely conserved, and the other amino acid residues involved in the anticodon recognition are conserved or replaced by functionally equivalent amino acid residues. The hydrogen bonds between ValRS and A35-C36 of tRNAVal clearly demonstrate how ValRS recognizes the major identity elements of tRNAVal.

Figure 3. A) Interactions between ValRS and the tRNAVal anticodon arm (stereoview).

Figure 3

A) Interactions between ValRS and the tRNAVal anticodon arm (stereoview). Each domain of ValRS is colored in the same way as in Figure 2. The trace of the C1' atoms of tRNAVal is shown as a yellow tube. The bases of tRNAVal are shown as sticks, where (more...)

The following A37 base is directed inward and stacks with G39 (fig. 3A). The orientation of A37 of tRNAVal may disrupt the stacking interaction between U32 and C33 within the anticodon loop, as described above (fig. 3A). Position 37 of tRNAVal is usually occupied by N6-methyladenosine (m6A).27 This small modification would not cause steric hindrance with any region of the tRNAVal anticodon loop. C38 is flipped out, and is bound to a pocket formed by Arg576, Asn580, and Ala6 (fig. 3A). The base of C38 is intercalated between the side chains of Arg576 and Asn580. Arg576 is either conserved or replaced by Trp, and Asn580 is strictly conserved. Position 38 is occupied by adenosine in E. coli tRNAVal (UAC). The previous kinetic analysis revealed that substitution of G for A38 reduces the aminoacylation efficiency of the E. coli tRNAVal 12-fold, but that of C or U does not affect the aminoacylation efficiency.13 The 2-NH2 group of G38 may cause steric hindrance with the pocket for position 38.

Interactions between the AS-Binding Domain of ValRS and the tRNAVal Anticodon Stem

The many interactions between ValRS and the tRNAVal anticodon loop may indirectly distort the structure of the tRNAVal anticodon stem, where the planarities of the C27•G43 and U28•A42 base pairs are impaired (fig. 3A). The distortion of these base pairs well explains previous imino proton NMR analysis (fig. 1B). Furthermore, the previous kinetic analysis of tRNAVal mutant showed that the stiffening of the anticodon stem by the introduction of five consecutive C•G base pairs cause a 50-fold decrease in the aminoacylation efficiency of E. coli tRNAVal.13 The distortion of the C27•G43 and U28•A42 base pairs in the T. thermophilus tRNAVal explains the importance of the anticodon stem flexibility. The anticodon stem of tRNAVal interacts with the AS-binding domain of ValRS.18 The first α-helix and the following loop of the AS-binding domain of ValRS form van der Waals interactions with the tRNAVal anticodon stem on the minor groove side (fig. 3A). There is no hydrogen bond between the AS-binding domain and the tRNAVal anticodon stem, while a positively charged patch on the surface of the AS-binding domain forms electrostatic interactions with the negatively charged phosphate groups of the tRNAVal anticodon stem (fig. 2B). The AS-binding domain of ValRS may function as a “splint” for the distorted anticodon stem. Actually, deletion of both the AS-binding and coiled-coil domains completely abolished the aminoacylation activity of the T. thermophilus ValRS,17 probably due to the lack of the interaction with the anticodon stem of tRNA.

Interactions of the C-Terminal Coiled-Coil Domain with the D- and TΨC-Loops of tRNAVal

The C-terminal coiled-coil domain also has a positively charged region (fig. 2B), which interacts with the “variable pocket”,17 a single-stranded protruding arch (comprising nucleotides 16, 17, 20, 59, and 60) formed by the D- and TΨC-loops22 (fig. 4). This interaction has been implied by the previous kinetic studies of yeast and E. coli tRNAVal mutants.13,23 In the present complex structure, A20 base is intercalated between the side chains of Arg843 and Leu815 (fig. 4). The N1 atom of A20 hydrogen bonds with the side-chain δH2 group of Asn847. The 2'-OH group of A20 hydrogen bonds with the NηH2 group of Arg818. The backbone phosphates of A20 and A21 form salt bridges with the NηH2 groups of Arg843 and Arg818, respectively. These two Arg residues are either conserved or replaced by Lys, while the Asn residue is not conserved. Correspondingly, nucleotide at position 20 is variable among tRNAVal species even from the same organism. Therefore, the hydrophilic interactions with position 20, rather than the specific recognition, are likely to be important. Furthermore, the G19•C56 tertiary base pair of tRNAVal interacts with the hydrophobic face formed by Pro833 and Val836, which are highly conserved in the ValRSs (fig.4). This hydrophobic interaction seems to stabilize the G19•C56 tertiary base pair, which associates the D- and TΨC-loops of the tRNA, together with the G18•Ψ55 tertiary base pair.

Figure 4. Interactions between the ValRS coiled-coil domain and tRNAVal (stereo view).

Figure 4

Interactions between the ValRS coiled-coil domain and tRNAVal (stereo view). The trace of the phosphorus atoms of tRNAVal is shown as a yellow tube. A20, U18•G55, and C19•G56 of tRNAVal are shown as a ball-and-stick representation, where (more...)

To investigate the role of the C-terminal coiled-coil domain, we performed mutational analyses of ValRS and tRNAVal(CAC) (18). We first analyzed a deletion mutant, Δ(795-862), which lacks the coiled-coil domain. The aminoacylation activity of the Δ(795-862) mutant was drastically reduced as compared with that of the wild type. The deletion of residues 795-862 not only increased the KM value for tRNA 30-fold, but also decreased the kcat value 20-fold. Subsequently, we analyzed an R818A/R843A double mutant, where Arg818 and Arg843 are replaced by Ala, to confirm the importance of the two salt bridges between the two Arg residues and the tRNAVal phosphate backbone. The R818A/R843A mutation affected the KM value for tRNA, but not the kcat value. The increase in the KM value by the R818A/R843A mutation was almost the same as that caused by the deletion of the coiled-coil domain. These results clearly show that the two Arg residues, Arg818 and Arg843, play a primary role in forming the stable ValRS•tRNAVal complex, and suggest that the other part of the coiled-coil domain, possibly the aforementioned G19•C56 binding pocket, is crucial for the catalytic processes, such as the correct positioning of the CCA end in the aminoacylation catalytic site.

On the other hand, we also analyzed tRNAVal mutants that either partially or completely lack tertiary interactions between the D- and TΨC-loops.17 If the D-loop-TΨC-loop interaction is stabilized by the coiled-coil domain, then the lack of the interaction should reduce the aminoacylation efficiency as well as the aforementioned ValRS mutations, Δ(795-862) and R818A/R843A. Actually, a point mutation at position 18 (G18U) and that at position 19 (G19C) increased the KM values for tRNA by about 10- and 20-fold, respectively. The aminoacylation activity of the G19C mutant could be recovered by the introduction of the C56G mutation (G19C/C56G), which retains the tertiary loop-loop interactions by a canonical Watson-Crick tertiary base pair. Therefore, we conclude that the tertiary loop-loop interactions are crucial for the efficient aminoacylation by ValRS. However, the partial disruption of the loop-loop interactions reduced the aminoacylation efficiency less than Δ(795-862). A drastic reduction in the aminoacylation efficiency was caused by the double mutation at positions 18 and 19 (G18U/G19C). The G18U/ G19C mutation increased the KM value 21-fold, and decreased the kcat value 30-fold. This reduction in the aminoacylation efficiency is comparable to that caused by Δ(795-862). The ValRS•tRNAVal•Val-AMS complex structure and these mutational analyses suggest that the reduction in the aminoacylation efficiency caused by Δ(795-862) is due to the absence of the tertiary loop-loop interaction of tRNAVal. The partial disruption of the tertiary interactions has been suggested by the previous footprinting analyses13 and by 19F and imino proton NMR spectroscopic analyses of tRNAVal and its complex with ValRS14,15 (fig.1). This partial disruption is probably prevented from extending to complete disruption by the interactions between the ValRS coiled-coil domain and the G19•C56 tertiary base pair of tRNAVal. These results imply that the coiled-coil domain of ValRS holds the partially disrupted tertiary base pairs, but not the completely disrupted base pairs, and subsequently maintains the canonical L-shaped structure of tRNAVal.

Interactions in the Acceptor Helix of tRNAVal

On the basis of kinetic analysis of tRNAVal mutants, the Horowitz group suggested that regular A-type RNA helix geometry of the acceptor stem, especially near the 4:69 base pair, is essential for the ValRS recognition.13 On the other hand, the Shimizu group reported, on the basis of identity switch experiment, that 3:70 and 4:69 base pairs in the acceptor helix are minor identity determinants.17 Furthermore, the 19F and imino proton NMR spectroscopic analyses of tRNAVal suggested that the environment around 3:70 and 4:69 base pairs is changed upon the complex formation with ValRS14,15 (fig. 1B). The present crystal structure shows no synthetase recognition of the acceptor stem, except for Arg385 whose NδH2 group hydrogen bonds with the O2' atom of C4. Nevertheless, the planarities of base pairs, 3:70, 4:69, and 5:68, are significantly distorted, probably due to the indirect effect of the complex formation, which is consistent with the results of NMR spectroscopy. Since the present complex structure represents “editing complex”, as described later, it provides no information of the recognition of the discriminator base by ValRS.

Viral tRNA-Like Structure

Turnip yellow mosaic virus (TYMV) has a 6.3-kb (+)-sense RNA genome whose 3'-terminal region, comprising 82 nucleotides, can fold into a structure that closely resembles the L-conformation of tRNAs. The 3'-terminal region has sequence similarity with tRNAVal, and is actually valylated by a plant ValRS,24-26 which is believed to be essential for the initiation of minus-strand replication in the viral life cycle. The Giegé group kinetically analyzed the recognition of the viral RNA with ValRSs from wheat germ and yeast, and found that the middle nucleotide of the anticodon and the 3' anticodon nucleotide contribute to the valylation activity, while the wobble position has no significant effect on valylation kinetics.24-26 Substitution of the discriminator base resulted in small decrease in Vmax/KM.24-26 Therefore, eukaryotic synthetases recognize the same identity elements in tRNAVal, as prokaryotic enzymes.

Amino-Acid Editing

Double-Sieve Amino-Acid Selection

The shape and the size of the L-threonine side chain are quite similar to those of L-valine, although one of the methyl groups of L-valine is replaced by a hydroxyl group in L-threonine. ValRS activates this isosteric L-threonine, and forms threonyl-adenylate (Thr-AMP) in addition to L-valine, as shown in Eq. 1. ValRS + L-threonine + ATP → ValRS•Thr-AMP + PPi (1)

Then, ValRS hydrolyzes Thr-AMP in a tRNA-dependent reaction (“pretransfer” pathway; Eq. 2). tRNAVal•ValRS•Thr-AMP→tRNAVal•ValRS + L-threonine + AMP (2)

When the Thr-AMP escapes the pretransfer editing, the Thr moiety is transferred once to the 3'-end of tRNAVal, and then the synthesized Thr-tRNAVal is hydrolyzed to Thr + tRNAVal by ValRS (the “posttransfer editing” pathway; Eqs. 3 and 4). tRNAVal•ValRS•Thr-AMP → Thr-tRNAVal•ValRS + AMP (3) Thr-tRNAVal•ValRS → tRNAVal•ValRS + L-threonine (4)

Fersht proposed the “double-sieve” (two-step substrate selection) model for the mechanism of the amino-acid selection by ValRS: L-threonine and L-valine are recognized by the shape in the first step, and L-threonine is discriminated from L-valine by the presence of the hydroxyl group in the second step (fig. 5).27-29 In addition to L-threonine, ValRS also activates and edits L-cysteine30,31 and an unnatural amino acid, L-α-aminobutyrate (α-But),9,28,30 which are smaller than L-valine by one methyl group.

Figure 5. Schematic drawing of the “double-sieve” concept for amino-acid selection by ValRS.

Figure 5

Schematic drawing of the “double-sieve” concept for amino-acid selection by ValRS.

The previous structure of T. thermophilus IleRS in a complex with L-isoleucine and L-valine revealed that the first sieve, which accommodates both L-isoleucine and L-valine, was identified on the aminoacylation domain, while the second, editing sieve, which is specific to L-valine, was found to exist on the CP domain, which protrudes from the aminoacylation domain.32 However, the aminoacylation catalytic site and the editing catalytic site are separated by about 35 Å, and it remains to be clarified how the valyl moieties of Val-AMP and Val-tRNAIle are transported from the aminoacylation site to the editing site.

The First, Size-Based Sieve

The Val-AMP analogue (Val-AMS), is tightly bound in a deep catalytic cleft of the central Rossmann-fold domain of ValRS, which serves as the first sieve in the double-sieve amino-acid selection (fig. 6).18 First, the α-NH3+ group of the aminoacyl moiety of Val-AMS provides three hydrogen bonds with the α-CO group of Pro42, the γ-COO- group of Asp81, and the γ-CO group of Asn44 (fig. 6A). Second, the aliphatic side chain of the valyl moiety fits into a hydrophobic pocket consisting of Pro41, Pro42, Trp456, Ile491, and Trp495 of ValRS (fig. 6A). The β;-CH group contacts both of the Trp456 and Ile491 side chains: the γ1-CH3 group contacts the Pro41 side chain, and the γ2-CH3 group contacts the Pro42 and Trp495 side chains (fig. 6B). It is impossible to fit an isoleucyl moiety into this pocket, because there is no room for the additional δ-CH3 group between the γ1-CH2 group and the Pro41 side chain (fig.6); the pocket of IleRS is wider and deeper than that of ValRS, mainly because of the replacement of the ValRS Pro41 by the IleRS Gly45. Therefore, the hydrophobic pocket of ValRS excludes L-isoleucine and larger amino acids, and snugly fits L-valine with respect to both its size and shape (fig. 6). Nevertheless, the isosteric L-threonine may fit into the pocket of ValRS (fig.6B). These features agree with the concept of the first, size-based sieve in the double-sieve mechanism of ValRS (fig. 5).29

Figure 6. A) Ball-and-stick representation of Val-AMP analog bound on the aminoacylation domain of ValRS (stereo view).

Figure 6

A) Ball-and-stick representation of Val-AMP analog bound on the aminoacylation domain of ValRS (stereo view). The catalytic KMSKS loop is colored in cyan. Blue dashed lines indicate hydrogen bonds between Val-AMP analog and ValRS. B) Contact-surface representation (more...)

Posttransfer-Editing Complex

In the ternary complex structure, the enzyme-bound tRNAVal has an L-shaped structure, like the enzyme-free tRNAPhe,33 with an angle formed by the acceptor and anticodon arms of about 90° (fig. 2). The 3' terminal strand of tRNAVal extends straight to the editing domain, and bound in the deep cleft formed by the protruding β-ribbon and the β-barrel structure (fig. 2).18 The extended 3'-terminal strand of tRNAVal contrasts sharply with the folded-back strand of tRNAGln in the aminoacylation complex of E. coli GlnRS34,35 (fig. 7A).

Figure 7. A) Two distinct conformations of the 3'-terminal strands of tRNAGln (blue) and tRNAVal (red) in the enzyme-bound state.

Figure 7

A) Two distinct conformations of the 3'-terminal strands of tRNAGln (blue) and tRNAVal (red) in the enzyme-bound state. The nucleosides in positions 73-76 are shown as plates. B) The tRNAVal CCA region is bound to the editing domain. The residues that (more...)

The 3'-terminal strand of tRNAVal is held primarily by interactions with the protein.18 The cytosine base of C74 contacts the side chains of Leu278 and Glu281, while the base and ribose moieties of C75 contact the side-chains of Glu261 and Phe264, respectively (fig. 7B). The 3'-terminal adenosine, A76, is more specifically recognized by the editing domain. The 5'-phosphate group of A76 forms a hydrogen bond with the side chain of Tyr337 (not shown). The adenine base of A76 is sandwiched between the side chains of Phe264 and Leu269 through van-der-Waals contacts (fig. 7C). The N1 atom and 6-NH2 group of A76 form hydrogen bonds with the α-NH and α-CO groups, respectively, of Glu261 (fig. 7C). These amino acid residues involved in the 3'-terminal A76 recognition are highly conserved among ValRSs. The base-specific recognition of A76 is consistent with the previous biochemical result that E. coli ValRS exhibited much lower hydrolytic editing activities with Thr-tRNAVal variants with the 3'-terminal A76 replaced by C, G, and U.36,37 Furthermore, the 2'-OH group of A76 forms a hydrogen bond with the γ-OH group of Thr214 (fig. 7C). Thus, the present complex structure is likely to represent the interaction of ValRS with tRNAVal at the posttransfer editing step for the Thr-tRNAVal hydrolysis.

The Second, Hydrophilicity-Based Sieve

In the complex of T. thermophilus IleRS and L-valine, the electron density of L-valine was observed not only on the Rossmann-fold aminoacylation domain but also on the editing domain.32 We recently solved 2.0-Å resolution crystal structure of the isolated editing domain of T. thermophilus IleRS in a complex with L-valine, which clearly shows that L-valine is recognized by invariant Asp328, His319, and Thr233 (not shown). When we modeled A76 of tRNAIle onto the editing domain, on the basis of the ValRS ternary complex structure, the covalent linkage of the α-CO group of the bound L-valine to the OH group of the modeled A76 moiety is allowed (not shown). Therefore, the valine-binding pocket near the A76-binding site on the IleRS editing domain is concluded to be for the posttransfer editing, to hydrolyze Val-tRNAIle.

On the other hand, in the T. thermophilus ValRS structure, Arg216, Thr219, Lys270, Thr272, Asp276, and Asp279 are near the A76-binding site, and form a more hydrophilic pocket (fig. 8),18 which spatially corresponds to the pocket for L-valine of the IleRS editing domain. These amino acid residues are strictly conserved in ValRSs. Then, we manually fitted L-threonine into the ValRS pocket, in a manner similar to the binding of L-valine to the posttransfer editing pocket of IleRS.18 The Asp279 side chain of ValRS protrudes into the pocket, as compared with that of the corresponding Asp328 residue in the T. thermophilus IleRS. In this model, the α-CO group of the L-threonine is covalently bonded to the 2'-OH group of A76, and the α-NH3+ group forms a hydrogen bond with the δ2-O atom of Asp279 (fig. 8). As for the side chain of the threonyl moiety, the γ-OH group also forms a hydrogen bond with the δ1-O atom of Asp279, while the γ-CH3 group is sandwiched between the β-CH2 of Arg216 and the ε-CH2 of Lys270. In addition, the γ-OH group may be recognized by the ValRS-characteristic Asp276. MALDI-MS identification of the binding sites for noncognate amino acids using the bromomethyl ketone derivatives suggested that the strictly-conserved His275, in neighborhood of Asp276, is involved in the editing reaction.38 A valyl moiety should be precluded from fitting into this pocket, because of the lack of proper hydrogen bonding with the δ-O atom of Asp279. In contrast, the aminoacyl moieties of cysteinyl-tRNAVal and α-aminobutyryl-tRNAVal may be accepted and hydrolyzed, because they are smaller than that of Val—tRNAVal by one CH3 group. Actually, a recent randomized selection experiment revealed that E. coli ValRS mutants that incorrectly charge tRNAVal with cysteine and/or aminobutyrate have mutations at Arg216 and Lys270, which constitute the putative threonine-specific pocket, as described above.39

Figure 8. Interactions of Thr-tRNAVal with the ValRS editing domain at the posttransfer editing step.

Figure 8

Interactions of Thr-tRNAVal with the ValRS editing domain at the posttransfer editing step. Left, a ball-and-stick representation (stereo view). The amino-acid residues constituting the Thr-binding pocket of the ValRS editing site and the bound tRNAVal (more...)

The present ValRS complex structure reveals that, at the posttransfer editing step, the second sieve of ValRS distinguishes L-threonine from L-valine based on the characteristic γ-OH group, whereas that of IleRS discriminates L-valine from L-isoleucine based primarily on the size. The comparison of the posttransfer editing sites between ValRS and IleRS demonstrates that a similar hydrolytic reaction accompanies the different manners of substrate selection. In both cases, the valyl moiety of Val-tRNAIle or the threonyl moiety of Thr-tRNAVal is transferred from the aminoacylation site to the 35-Å remote editing site by a simple bending of the 3'-terminal acceptor strand of tRNA, analogous to the editing by DNA polymerase, as a “shuttling mechanism” proposed by the ValRS ternary complex structure18 and by the crystal structure of Staphylococcus aureus IleRS complexed with E. coli tRNAIle and the antibiotic mupirocin.40


The history of investigations, the mechanism of tRNA recognition, and the amino-acid selection mechanism of ValRS have been overviewed. The recent crystal structure of T. thermophilus ValRS complexed with tRNAVal and Val-AMP analog explains the substrate recognition mechanism and suggests the editing reaction mechanism. However, more precise mechanisms of substrate recognition and aminoacylation/editing reaction await structure determination of aminoacylation complex at higher resolution. The mechanism of tRNA and amino-acid recognition by ValRS quite resembles that by IleRS, which definitely shows that ValRS and IleRS are closely-related in their evolution.


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