Idiosyncrasy and identity in the prokaryotic phe-system: Crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP

Abstract

The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)₂ heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N-terminus, A1 and A2 belong to the α-subunit and B1-B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix-bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil-short helix structural fragments. The N-terminal domain of the α-subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N-terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA^Phe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ∼5 Å is required for C-terminal domain B8, and of ∼6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.

Introduction

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes that covalently attach amino-acids to their cognate tRNAs. Based on a comprehensive analysis of their sequences and 3D-structures, aaRSs were partitioned into two classes.^1,2 The partition correlates with differences in topologies of the catalytic domains. Class I aaRSs contain so-called “Rossmann dinucleotide binding fold,” whereas class II enzymes possess antiparallel β folds. The class II catalytic domains share three signature motifs; motif I has a helix-turn-strand organization and it is a part of an oligomerization interface, whereas residues from motifs II and III participate in the activation of cognate amino acids and subsequent aminoacylation of the tRNA.^1–4

The two classes of aaRSs are also distinguished by the stereochemical modes of attack on the carbonyl of the aminoacyl-adenylate. Class I aaRSs attach amino-acids to the 2'—OH group of ribose at the 3′-end adenosine of tRNA, while class II aaRSs attach them to the 3'—OH group. Phenylalanyl-tRNA synthetase (PheRS) is the only exception to aaRSs family in this matter; its active site has structural organization as all class II enzymes, while attaching phenylalanine to the 2'—OH of terminal ribose of the tRNA^Phe.^5,6 Structural and phylogenetic analysis reveal three different forms of PheRS: (a) bacterial heterotetrameric (αβ)₂;^7–10 (b) eukaryotic/archaeal heterotetramic (αβ)₂;^10,11 and (c) mitochondrial monomeric.^12,13

Erroneous activation and subsequent misaminoacylation due to close resemblance of some cognate and noncognate amino acids become a risk to the cell's viability. For that reason quality control was developed by aaRSs when the active site is distinguishing cognate from noncognate amino-acids with great accuracy. If, nevertheless, the noncognate amino-acids or any other natural compounds are activated by aaRSs, an additional step of quality control (editing mechanism) is invoked, hydrolysing the misactivated aminoacyl adenylate (pretransfer editing) or the mischarged aminoacyl-tRNA (aa-tRNA) (post-transfer editing).¹⁴

Although considerable progress has been made in the last few years with respect to understanding the mechanisms of quality control, the idiosyncrasies of the aaRSs, and PheRSs belonging to different kingdoms in particular, have hampered efforts to provide a more detailed overall picture of function. This is especially true with regard to the structural organization of editing domains, mechanisms of substrate discrimination and hydrolysis, pathways of signal transduction and tRNA translocation from synthetic to editing site. Early biochemical experiments showed that yeast PheRS possesses an editing activity towards the noncognate amino acid tyrosine (Tyr).^15,16 It was proposed that the pretransfer hydrolysis triggered by tRNA^Phe makes a major contribution to Tyr rejection. Later it was shown that EcPheRS also misactivates Tyr and further corrects this error at the post-transfer stage.^17,18 Based on structural studies and genetic screen, a few residues of the EcPheRS β-subunit (B3-B4 domains) presumably associated with the editing activity of the enzyme were identified.^8,17,19 Though some kinetics and biochemical data regarding the editing activity of EcPheRS were published,^17,18 the structure of the enzyme has not yet been solved. Here, we report the first crystal structure of the E. coli phenylalanyl-tRNA synthetase. The crystal structure of EcPheRS contributes to our knowledge of aminoacyl-tRNA synthesis and provides new structural facets of phenylalanylation reaction and editing activity. In view of its complexity each PheRS structure reveals novel idiosyncratic characteristics in the architecture of the synthetic and editing sites, along with intersubunit and quaternary organizations.

Results and Discussion

Overall structure of EcPheRS and comparison with other class IIc aaRSs

The X-Ray crystal structure of the EcPheRS was determined at 3.05 Å resolution [Fig. 1(A)]. The crystals belong to space group P2₁2₁2₁ with one heterotetrameric EcPheRS molecule in the asymmetric unit. The molecular replacement (MR) method was implemented for structure determination. The atomic coordinates of the TtPheRS structure (PDB code 1PYS⁸) were used to construct a preliminary model and to carry out an initial search. The refined structure yielded a crystallographic R_work = 23%, and R_free = 28%. At 3.05 Å resolution 96% of ϕ and ψ angles lie in the most favored and allowed regions of the Ramachandran plot. Crystallographic data collection and structure refinement statistics are presented in Table I.

Figure 1.

Structures of PheRS heterotetramers. (A) Structure of the (αβ)₂EcPheRS; Structural domains: the A1/A2 domains that form CAM (residues 108-307) and N-terminus that forms triple helix (TH) belong to α-subunit and colored in green and blue respectively; the B1-B8 domains belong to the β-subunit. Domains B6/B7 form CLM (residues 483-691) are colored in red. The structural domains associated with the symmetry related heterodimer (colored in the same colors) are asterisked. (B) The active site of EcPheRS complexed with an aminoacyl-adenylate; (C) Schematic representation of two heterotetrameric molecules depicted as a cylinders: EcPheRS and TtPheRS. The intramolecular twofold axes a and b relate αβ heterodimers in heterotetramer (αβ)₂; (D) The central four-helix bundle interface of TtPheRS; (E) The central four-helix bundle interface of EcPheRS. The CLMs have shorter helices as compared to TtPheRS; (F) Intramolecular interactions between CLMs and CAMs at central interface. Interactions between charged residues exposed to this area are presented. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Table I.

Crystallographic Data and Refinement Statistics for EcPheRS^a

Data collection
Wavelength (Å)	0.9762
Space group	P212121
No. molecules in asymmetric unit	1
Cell dimensions
a, b, c (Å)	65.547, 178.936, 254.417
α, β, γ (°)	90,90,90
Resolution (Å)	40–3.05 (3.16–3.05)
Rmerge(%)	0.09 (0.66)
I/σ	15.5 (1.55)
Completeness (%)	99.3 (99.3)
Redundancy	3.6 (3.4)
No. of reflections (total/unique)	201,437/56,171
Refinement
Resolution range (Å)	40–3.05
No. unique reflections	56,171 (5503)
Rwork/Rfree (%)	23/28
Average B-factor	79.8
Number of protein atoms	16,710
R.m.s.d.'s
Bond lengths (Å)	0.013
Bond angles (°)	1.813

^aValues in parentheses are for highest-resolution shell.

The EcPheRS is a (αβ)₂ heterotetramer, built of two αβ heterodimers [Fig. 1(A)]. Each αβ heterodimer consists of 11 structural domains; three of them belong to α-subunit: N-terminal domain, A1 and A2, while the B1-B8 domains belong to the β-subunit. The core of the EcPheRS is formed by two catalytic modules (CAMs) (residues 108-307) from the α-subunit, and two catalytic-like modules (CLMs) (residues 483-691) from the large β subunit [see also legend to Fig. 1(A)].

EcPheRS structure was compared with other prokaryotic and eukaryotic PheRS structures including the TtPheRS,^8,9,19–21HcPheRS,¹⁰ and HmPheRS.¹² The structural alignment between the heterotetramers TtPheRS and the EcPheRS model indicates that there is a difference in orientation of the intramolecular axes connecting the dimers. For a given pair of enzymes, the angle between the two intramolecular axes is 3.5° [Fig. 1(C)]. When the structure of EcPheRS is compared with that of HcPheRS, the angle between the two intramolecular axes is 7.1°.

Two insertions 157-173 and 294-302 in the sequence of TtPheRS α-subunit as compare to EcPheRS contribute to additional intramolecular interactions. They more efficiently protect the four helix bundle interface from the solvent at high temperature environment, characteristic for thermophiles.

The differences between the α-subunit of EcPheRS and HmPheRS basically associated with the N-terminal region of the subunit and with one large insertion in HmPheRS.¹² Superimposition of the catalytic core of EcPheRS onto that of HcPheRS displays differences between the N-terminal regions of the α-subunits. Another remarkable distinction is the B2 domain, which is missing from the human cytosolic β subunit.¹⁰

The (αβ)₂ subunit communication

The multimerization area of EcPheRS created by αβ-heterodimers, lies along the intramolecular twofold axis just as in other cytosolic class IIc aaRSs. Helices forming the central part of four-helix bundle interface in EcPheRS are the structural elements of motif 1 in both α and β subunits.^7,8 Core of the TtPheRS's (αβ)₂ interface looks quasi-tetrahedral and comprises four-helix bundle with an interhelical separation of 14 Å.^7,8 Positively and negatively charged residues presented to interface by four subunits fully compensate each other and total charge within this area equals zero. The clear-cut distinction derived from superimposition of two structures is that motif 1 helix in the EcPheRS belonging to CLM (i.e., to β-subunit) is much shorter than its partner in TtPheRS. It consists of only three α-helical turns followed by β-strand. Therefore, the structural core of EcPheRS comprises two short helices of CLMs followed by β-strands and by two helices of CAMs [Fig. 1(D,E)]. Nevertheless, the intramolecular interactions formed by side chains exposed to the interface are preserved and the total charge within this area is also zero, thereby stabilizing the whole architecture of the heterodimer: Gluβ490 from CLMs interacts with Argα115 from CAMs, while Hisβ488 from CLMs interacts with Gluα122 from CAMs [Fig. 1(F)].

Active site loops

Three loops are shown to be involved in closing up the active site of EcPheRS in a manner analogous to that in TtPheRS: a motif 2 loop, a “helical” loop, and FPF loop. The motif 2 loop (residues 194-205 of the α-subunit) has a conformation similar to that of TtPheRS, whereas in HmPheRS it is shifted by 2.5 Å toward the center of the active site cavity. The second, helical loop (residues 145–158 of the α-subunit), also has a conformation similar to that of TtPheRS. This loop undergoes conformational changes upon substrate binding. The backbone of the third FPF loop, which envelopes the active site (residues 246–254 of the α-subunit), shows a remarkably stable conformation. A specific recognition of the phenylalanine is achieved by interactions of the substrate phenyl ring and the two neighboring phenyl rings of Pheα248 and Pheα250 from motif 3; these contacts are arranged in ‘edge to face’ manner with the phenyl ring of the substrate.²¹

The N-terminus of the α-subunit

The N-terminus of the α-subunit plays an important role in binding of the tRNA^Phe in the bacterial phe-system. In the native structure of the TtPheRS, as well as in the complexes with small substrates (Phe, Phe-AMP, Tyr, etc.), the first 84 residues of the N-terminus in the α-subunit are disordered. Only in the presence of tRNA, the N-terminal region can be seen in the electron density maps and shaped into a coiled coil. However, first 15 residues at the N-terminus were not clearly visible, and thus not included into the final model.^8,9

Contrary to TtPheRS, the N-terminus of the EcPheRS in apo-form is clearly seen in the electron density map and displays a triple helix motif [Fig. 2(B)]. Whether the observed triple-helix changes its conformation to the coiled-coil structure upon tRNA^Phe binding, remains unclear. Looking for specific residues in the N-terminal region that could contribute to RNA-protein interactions, we found that the N-terminal region of the α-subunit contains a GXXG motif (YLGKKG in EcPheRS) highly conserved among prokaryotic PheRSs (see multiple sequence alignment for α-and β-subunits presented in Supporting Information). In TtPheRS complexed with tRNA^Phe, only one out of two Lysine residues (K28) makes a direct and nonspecific contact with O2P of Ade43, while the K29 is exposed to solution. We suggest that first lysine in this motif from EcPheRS play similar role generating electrostatic interactions with phosphodiester backbone of tRNA [see Fig. 2(C)]. Interestingly, GXXG motif revealed also in other RNA-binding proteins, structurally not related to aaRSs (KH and CRM domains) and associated with RNA-binding activity.^22–24

Figure 2.

The superimposition of the catalytic core of the TtPheRS-tRNA complex (red) onto that of EcPheRS (green) is evidence for existence of the conformational distinctions between two structures: (A) the displacement of B2 domain by approximately 18–20 Å; (B) the displacement on 7–8 Å of the N-terminus of the α-subunit. A triple helix of EcPheRS and coiled-coil of TtPheRS are depicted; (C) The anticodon loop of tRNA (orange) is clamped between the B8 and the N-terminus of the α-subunit. Conformational changes, which may occur in EcPheRS upon tRNA binding. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The active site: a phenylalanyl-adenylate binding pocket

The amino-acid binding pocket of EcPheRSs is a deep cavity within the active site, similar to that observed in TtPheRS⁸ and in the HmPheRS.¹² Analysis of EcPheRS superimposed onto CAM from the TtPheRS-PheAMP complex shows similarity of Phe and AMP binding modes in two enzymes. The phenyl ring of the substrate is stabilized by aromatic-aromatic interactions with two conserved residues: Pheα248 and Pheα250 [Fig. 1(B)]. The α-NH₃⁺ of Phe-AMP makes hydrogen bonding with Serα171, Glnα169, and Gluα210. The AMP moiety is sandwiched between Pheα206 and hydrophobic part of Argα301 of the α-subunit.⁹ Metα148 and Trpα149 are two key residues of the helical loop in TtPheRS that control productive binding of the tRNA.²⁰ The side chain of Metα148 (Aspα154 in EcPheRS) opens or closes a barrier for placement the CCA end of tRNA^Phe within the active site.²⁰ While Trpα149 (Hisα155 in EcPheRS) participates in stabilization of the negatively charged carbonyl oxygen of the substrate, it is oriented perpendicular to the base of A76 and forms a hydrogen bond with it. Most of the residues cooperating in the binding and recognition of the Phe-AMP and the A76 are invariant in all PheRSs. The helical loop is an exception to this rule. Not only because of the mutations in this region, but due to changes of its overall conformation as well. It should be noted that residue Trpα149 (TtPheRS) is replaced by Hisα155 in EcPheRS and as such is also capable of making interactions analogous to those formed by Trp.

It is notable that substitution of Hisα179 in TtPheRS for Gluα169 in E. coli enzyme leaves hydrogen bonding interaction with the first intermediate unaffected. Thus, we may conclude that the net of interactions of Phe-AMP within the active sites in EcPheRS resembles this found in TtPheRS.

Editing site

The editing site of PheRS is localized ∼35 Å from the active site, at the interface of the B3/B4 domains of the bacterial PheRS.¹⁹ The B3 domain resembles the α-β “sandwich” with a layer of two α helices packed against a four-stranded antiparallel β sheet. The largest insertion (residues 265–328) constitutes domain B4. The crystal structure of the TtPheRS complexes with noncognate amino acid Tyr revealed that two Tyr molecules bind within the editing and synthetic sites. Moreover, kinetic experiment showed that the Tyr molecule can be activated by PheRS and transferred onto the tRNA^Phe.^17,19 Thus, Tyr loaded on tRNA^Phe must be transferred from the synthetic to the editing site, in order for hydrolysis to occur (the so-called, “translocation step”).^14–17

The architecture both of the synthetic and editing sites enables binding of aromatic moiety of Tyr. The phenyl ring of Tyrβ360 (Pheβ360 in TtPheRS) and the side chain of Proβ263 (Proβ259 in TtPheRS) promote “edge-to-face” interactions with Tyr. Pheβ360 is strictly conserved in eubacterial PheRSs while, in several cases, Proβ259 is replaced with Ile or Leu, capable of participating in hydrophobic interactions. The anchoring of the OH group of Tyr is achieved by its interactions with the Oɛ1 of Gluβ334 and the main chain amide of Glyβ315.¹⁹ A remarkable structural peculiarity of the editing site is the appearance of the invariant hydrophilic Gluβ334 within the fully hydrophobic environment formed by Tyrβ216, Glyβ218, Pheβ267, Ileβ304, Glyβ314, Glyβ315, Alaβ336, and Pheβ338 (according to EcPheRS nomenclature). Thus, Gluβ334 in EcPheRS also plays a critical role in specific recognition and discrimination of the Tyr moiety.

The B2 domain rearrangement

Superposition of TtPheRS onto EcPheRS shows similar arrangement of the B3/B4 domains, with a considerable shift of 18–20 Å observed for the B2 domain. This domain is a structural homologue of the EMAPII/OB fold, which has been shown in other systems to contribute to tRNA binding. In the TtPheRS-tRNA^Phe complex, no direct interactions were detected between the B2 and the tRNA molecule. The B2 domain is connected to the central part of the enzyme via two strands. In EcPheRS the outstanding mobility of B2 revealed by 20 Å displacement as compared to its position in TtPheRS. Different orientation of B2 bears witness to the fact that the domain may be considered as a secondary tRNA-binding region by promoting the translocation of the mischarged tRNA from synthetic to the editing site [Fig. 2(A)].²⁵

Modeling of the EcPheRS: tRNA complex

Modeling of the EcPheRS-tRNA complex was generated using superposition of TtPheRS-tRNA^Phe complex onto the EcPheRS. The acceptor stem and the CCA end of tRNA^Phe are localized within the area of the active site of the α-subunit and structural domains B1, B3, and B7 from the same αβ heterodimer. Analysis of EcPheRS superimposed onto CAM from the TtPheRS-tRNA complex shows that tRNA in the EcPheRS α-subunit will have a conformation similar to that of TtPheRS. The CCA interacts with residues Hisα202, Argα301, Pheα206, Hisα155, and Aspα154 (according to EcPheRS) of the α-subunit, which are in the same conformation as that of TtPheRS. The anticodon arm and the variable loop of the tRNA are clamped between the B8 domain and the N-terminal helical arm of the α-subunit from the symmetry-related heterodimer. Residues Serβ755, Aspβ742, and Tyrβ744 of the B8 domain are candidates for interactions with the anticodon triplet of tRNA^Phe.

Analysis of EcPheRS superimposed onto the CAM of the TtPheRS-tRNA complex shows that the B8 domain of EcPheRS along with the N terminus are positioned in an “open” conformation, and may switch to a “closed” conformation in the presence of tRNA. In a closed state the B8 domain and the N-terminus of α-subunit have to be displaced by 4–5 Å and of 6–7 Å, respectively. Formation of the complex may also be accompanied by conformational transition from a triple helix to a coiled-coil structure [Fig. 2(B,C)]. Coiled-coil and B8 domain can move toward each other, thus clamping the anticodon arm and the variable loop of the tRNA.

Conclusion

We have determined the crystal structure of E.coli PheRS complexed with phenylalanine and AMP. The CLMs of EcPheRS present to the central part of the interface the helices, which are substantially shorter, than those in TtPheRS. The long helices effectively converted to the helix-coil supersecondary structures and as a consequence, central interface in EcPheRS changes its architecture, while keeping the electrostatic charge within this region close to zero [see Fig. 1(F)]. Thus, knowledge of EcPheRS structure prompt us to extend our speculation about the structural organization of the core interface area in heterodimeric PheRSs: apparently characteristic four-helix bundle is not the prerequisite of the central core interface topology in (αβ)₂ aaRSs. This in turn revises our understanding of the role that motif 1 plays in heterodimeric structure formation. An intriguing question is related to the understanding of the structural and functional role of CLM. This module is likely to carry out dual functions, shared between domains B6 and B7. Firstly, both participate in nucleation of the αβ interface, generating a vast area of hydrophobic contacts with the convex side of CAM β-sheet. Secondly, insertion domain B7 topologically resembles insertion domain A2 with minor exceptions. As it appears, such a similarity is related to the additional structural stability that B7 and A2 contribute to heterodimer formation: they create one, common to both subunits, four-stranded antiparallel β-sheet. Together with the symmetry mate (molecular twofold axis) two β-sheets protect the interface region and the cavities of the active sites. It is worth of mentioning that two insertions in TtPheRS 157-173 and 294-302 also serve for additional protection of the central interface, accounting for that a given organisms can thrive and function at relative high temperatures.

The remarkable plasticity of the synthetic active site and the structural diversity of PheRSs in different kingdoms made these enzymes and, EcPheRS in the first place, of considerable promise instrument for introduction of non-natural amino acids into the protein polypeptide chains. Indeed, EcPheRS was among the first aaRSs used to introduce Phe analogs into proteins. Ibba et al.²⁶ have demonstrated an attachment of the para-halogenated Phe analogs to tRNA and their in vivo incorporation into cellular proteins by using E. coli Ala294Gly mutant (Ala314 in T. thermophilus PheRS) that exhibits relaxed substrate specificity. Further studies revealed that phenylalanine analogs substituted with various chemical groups (bromo-, iodo-, ethynyl-, cyano, and azido-) at para position were efficiently incorporated into recombinant proteins by the mutant PheRS.^26,27 However, this mutant failed to sufficiently incorporate p-acetylphenylalanine into the proteins. The analysis of EcPheRSs 3D-sructures identified another mutation that may be crucial for plasticity and active site specificity, the Thr251Gly (Val261 in TtPheRS). Indeed doubly mutated PheRS (T251G, A294G) as predicted by the design algorithm efficiently incorporates in vivop-acetylphenylalanine into recombinant protein.²⁸ The net result of these active-site mutations is formation of the space in the phenylalanine binding pocket needed to accommodate sterically demanding para-substituted analogs. Interestingly the relaxed specificity of these mutants appeared to be directed mainly to the para-position of the amino acid substrates. It is notable that from structural point of view appearance of relaxed specificity in EcPheRS clearly correlates with mutation Thr251Gly: a given mutation enlarges the size of amino acid binding pocket and by doing so it is possible to place in this area amino acids with bulkier side chains. Less obvious is the emergence of relaxed specificity in response to mutation Ala294Gly. At the first glance there are no steric hindrances between para-substituted analogs of phenylalanine and side chains of amino acids lining the walls of amino acid binding pocket. However, natural plasticity of the amino acid binding pocket in TtPheRS allows p-Cl-Phe to be bound and recognized by the wild-type enzyme.⁹ For tackling this question the structures of mutant variant complexed with various ligands, have to be determined. All this suggests that engineered specificity for one enzyme may be considered as a natural one for another representative in the family.

The 3D-structure of the EcPheRS provides insight into the structural basis of many biochemical experiments carried on the EcPheRS. Structural evidence will clarify the aminoacylation, translocation and editing mechanisms in different pathogenic PheRSs. Due to the essential role of aaRSs in biosynthetic machinery and thus, in cell viability, inhibiting of their enzymatic activity is detrimental to the cell and may provide the basis for developing a novel antibacterial agents. The major observation which follows from our results is that the modes of binding and of the recognition of cognate tRNA^Phe are different in prokaryotes and eukaryotes. These sharp biological distinctions suggest that PheRS holds not only a unique position among the other aaRSs in terms of complexity but also make the enzyme of considerable promise for creation of new antibacterial agents. High-resolution crystallographic structures of inhibitors with the PheRS from various pathogenic sources would enable characterizing the parameters dominating the affinities of inhibitors as well as their binding modes.

Methods

Protein expression, purification, and crystallization

The EcPheRS operon as found in the E. coli genome was cloned into His-tag PQE31+ vector (Agilent Technologies) and overexpressed in E. coli strain XL1 blue (Agilent Technologies). Substitution of PheRS E. coli methionines to selenomethionines (Se- PheRS E. coli) has been done as described in Van Duyne et al.²⁸EcPheRS protein was purified and concentrated up to 10 mg/mL in solution P (20 mM Tris–HCl buffer pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM MgCl₂, 10% glycerol, 1 mM Phe and 1 mM ATP). The EcPheRS was crystallized by the hanging-drop vapor-diffusion method at 20°C using 4 μL drop. The protein solution was mixed at 1:1 ratio with the precipitant containing 17–20% PEG 8000K, 0.2 M MgCl₂, 0.1 M Tris–HCl buffer (pH 8.5), 3% 1,6 Hexandiol, and 3% d-galactose. The crystals appeared within 3 days. The Se-Met substituted crystals were grown in an analogous way.

Data collection and processing

For data collection at cryogenic temperature (100 K), the crystals were transferred to a mother-liquor solution containing 20% (v/v) ethylene-glycol, mounted on a cryogenic loop and flash-cooled using an Oxford Cryostream low-temperature device. Data were collected from shock-frozen crystals using synchrotron radiation beam at ID23-1/ID14-4, ESRF, France. The crystal diffracted to 3.05 Å resolution. The crystals belong to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 65.5, b = 178.9, c = 254.4, and α = β = γ = 90°. Data processing was performed with HKL-2000²⁹ (Table I). The molecular replacement method has been used for solving 3D-structure of E. coli PheRS. To determine whether the orientation of the search model within asymmetric unit was unambiguous, three different programs MOLREP,³⁰ PHASER³¹ and AMORE³² programs were invoked. All solutions were closely similar in appearance. Since anomalous data set was collected at 6 Å only, we didn't apply it for initial phasing. However, we used Se-Met residues as anchoring points at the stage of electron density maps interpretation. Positions of selenium atoms were located from the anomalous difference Fourier map (Δano), with the phases derived from the partial MR model. Refinement of Se atoms positions and calculation of an experimental map from the MAD dataset was accomplished by means of the SHARP program.³³ Anomalous different map was calculated by FFT and SIGMAA. Combination of experimental and molecular replacement phases was done with MLPHARE³⁴ and SIGMAA³⁵ programs. Model building and refinement was performed using the COOT,³⁶ CNS package³⁷ and REFMAC.³⁸ The subsequent refinement procedures: conjugate-gradient energy minimization, combined simulated annealing and B-factor refinement were then carried out. For free R-factor calculation, a random 5% of the data was omitted during refinement.

The coordinates have been deposited in the Protein Data Bank under PDF ID Code 3PCO.

Glossary

Abbreviations:

aaRS

aminoacyl-tRNA synthetase

Ec

Escherichia coli

Hc

human cytosolic

Hm

human mitochondrial

PheRS

phenylalanyl-tRNA synthetase (other specific aminoacyl-tRNA synthetases are denoted by their three-letter amino acid designation)

Tt

Thermus thermophilus.