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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochimie. Author manuscript; available in PMC Mar 1, 2012.
Published in final edited form as:
PMCID: PMC3039092
NIHMSID: NIHMS261497

Structure of Leishmania major Methionyl-tRNA Synthetase in Complex with Intermediate Products Methionyladenylate and Pyrophosphate

Abstract

Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg2+ ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of E. coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNAMet complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.

Keywords: aminoacyl-tRNA synthetase, protozoa, drug target, leishmaniasis, trypanosomiasis, enzyme product complex

1. Introduction

A spectrum of diseases called leishmaniases, with outcomes ranging from debilitating disfiguration to death, is caused by multiple species of Leishmania parasites. These unicellular eukaryotic organisms, belonging to the order of Kinetoplastida and family of Trypanosomatidae, are transmitted by certain species of sand fly. It is estimated that there are two million new cases each year in the tropics and subtropics with over 350 million people at risk worldwide [1, 2]. Current anti-leishmania drugs have serious shortcomings as evidenced by miltefosine, the only oral drug against visceral leishmaniasis, which is teratogenic [3]. Moreover, resistance against existing drugs is increasing and there are no effective vaccines [4]. The need for novel effective drugs for treatment of leishmaniases is therefore urgent. The same can be said for diseases caused by other closely related trypanosomatid species such as Trypanosoma brucei, the causative agent of sleeping sickness, and Trypanosoma cruzi, responsible for Chagas disease [5-7], and it is hoped that information obtained about key proteins from any of these parasites will benefit drug design efforts against the other species.

A major goal of the Medical Structural Genomics of Pathogenic Protozoa collaboration is to identify and characterize potential drug targets in several eukaryotic pathogens, including the kinetoplastids, to provide a platform for anti-protozoan drug development [8]. In this pursuit, we selected the aminoacyl-tRNA synthetases (aaRS) for structural and biochemical characterization [9-12]. AaRS are essential to the core biological process of translating nucleotide-encoded gene sequences into proteins and are thus present in all organisms spanning all three domains of life. They are grouped into two classes based on structural and sequence features of the catalytic core [13-19]. Both classes are further divided into subclasses based on additional commonalities of sequence motifs and structural elements [18]. Very generally speaking, each aaRS recognizes a single amino acid and attaches it to a tRNA whose anti-codon matches a codon for that amino acid, but exceptions to these rules are prevalent and many aaRS additionally rely on various proofreading or editing mechanisms to ensure faithful translation of the genetic code. To charge a tRNA, the aaRS must catalyze at least two reactions. The amino acid is first activated with ATP to form an aminoacyl-adenylate intermediate and then is transferred onto the terminal adenosine of the tRNA [18]. Interference with any step throughout this process will inhibit the formation of properly charged tRNA leading to disruption of normal protein chain elongation during translation.

Consistent with the crucial role of aaRS in translation, studies in various organisms have shown these enzymes to be essential. Most eukaryotes carry two genes for each aaRS (cytoplasmic and mitochondrial) but these cannot compensate for each other when one is knocked down. Surprisingly, trypanosomatids appear to harbor only one aaRS per amino acid except for AspRS, LysRS and TrpRS [5-7]. RNA interference experiments have shown that each of the two T. brucei TrpRS enzymes are essential [20] and that knockdown of the single T. brucei HisRS [10], IleRS (unpublished), or MetRS (manuscript in preparation) leads to complete growth arrest in the bloodstream forms of this protozoan. As a general rule, these enzymes are required for cell survival and consequently are excellent drug targets from a functional perspective. However, because of their conservation in all living organisms, the ability to selectively inhibit the pathogen enzyme over the host enzyme remains a challenge. This underscores the utility of incorporating structural knowledge of the enzyme into the drug design process.

Despite this challenge, aaRS have been successfully targeted by selective inhibitors. A prime example is the drug mupirocin, targeting prokaryotic IleRS [21]. It is used topically to treat bacterial infections including methicillin-resistant Staphylococcus aureus (MRSA). Amazingly, selectivity over host homologs has been attributed to differences in only two residues at the active site [22]. Also, a lead compound (SB-425076) targeting S. aureus MetRS [23-26] was further developed into an analog (REP8839) that shows antibacterial activity against several Gram-positive bacteria and reached clinical trials [27-30]. Another related MetRS-specific diaryldiamine compound (REP3123) has shown promising results in preclinical studies against Clostridium difficile [31-33]. This series of MetRS inhibitors, which are methionine- but not ATP-competitive, shows that selective inhibition of pathogen MetRS is achievable.

We report here the 2.0 Å crystal structure of MetRS from L. major (LmMetRS) in complex with two products, L-methionyladenylate (MetAMP) and Mg-pyrophosphate. MetRS belongs to aaRS subclass Ia, which contains a catalytic core comprised of a Rossmann fold with a connective peptide insertion divided into two contiguous parts named CP1 and CP2, the two characteristic Class I aaRS sequence motifs HIGH and KMSKS (KISKS in L. major), the stem-contact-fold (SCF), and a helical bundle (Fig. 1). The present structure is the first class I aaRS to be captured as a complex with the two intermediate products methionyladenylate (MetAMP) and pyrophosphate, along with a bridging Mg2+, in active site. It has the most compact active site observed in MetRS structures to date, displaying large conformational changes compared to other MetRS structures, particularly in the KISKS loop and in CP1, that are attributed to the presence of the pyrophosphate. Importantly, there are several amino acid differences near the methionine and adenosine binding pockets of LmMetRS compared to even the closest human homolog, the mitochondrial enzyme. This holds promise for obtaining inhibitors with higher affinity for the parasite than the human enzymes.

Figure 1
Crystal structure of the catalytic core of L. major methionyl-tRNA synthetase

2. Materials and methods

2.1. Protein expression and purification

The catalytic core (residues 206-747) of the 747 residue MetRS from L. major (GeneDB identifier LmjF21.0810) was PCR amplified from genomic DNA of L. major strain Friedlin and cloned into E. coli expression vector AVA0421. This catalytically active N-terminal truncation mutant was pursued for structural studies after significant effort was expended attempting to crystallize the full-length protein to no avail. Protein was purified by a Ni-NTA affinity column followed by size exclusion chromatography (SEC) on a XK 26/60 Superdex 75 column (Amersham Pharmacia Biotech) using SGPP standard buffer (20 mM HEPES, 0.5 M NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 5% glycerol, 10 μM ZnCl2, 0.025% NaN3 at pH 7.0) [34]. The peak SEC fractions containing the target protein were pooled and spiked with 10 mM Mg-ATP, 10 mM L-methionine and 10 μM ZnCl2. The protein sample was concentrated to 24 mg/ml using an Amicon concentrator. Purified protein retained a 22 amino acid residue expression tag composed of an N-terminal His6 tag and a 3C protease cleavage site.

2.2. Protein crystallization

Purified LmMetRS protein was screened by sitting drop vapor diffusion for crystallization leads using the Phoenix crystallization robot (Art Robbins Instruments) and the JCSG+ Suite sparse matrix crystallization screen [35] (Qiagen). Several leads were further optimized to yield crystals suitable for X-ray diffraction data collection. The best crystals were obtained by vapor diffusion from hanging drops equilibrated at room temperature against a reservoir containing 0.2 M potassium formate or potassium nitrate (pH 7-7.5) and 24-28% PEG 3350. These crystallization drops consisted of 2 μL protein at 22 mg/mL plus 2 μL of the reservoir solution. The protein was in a buffer consisting of 25 mM HEPES pH 7.0, 0.5 M NaCl, 0.025% sodium azide, 5% glycerol, 1 mM TCEP, 0.01 mM ZnCl2, 10 mM Mg-ATP and 10 mM L-methionine. 1 mM TCEP and 10 mM L-methionine were additionally added to the protein solution prior mixing with the reservoir solution. Crystals diffracting up to 2.0 Å resolution grew in two days at room temperature.

2.3. Data collection and structure determination

LmMetRS crystals were screened at the Stanford Synchrotron Research Lightsource (SSRL) on beamlines 9-2 and 11-1 using the SSRL automated mounting (SAM) system [36]. Data were collected at 100 K and a wavelength of 0.979 Å on a MarMosaic-325 CCD detector using the Blu-Ice software package [36]. 130° of data with 1° oscillation/image were collected. Data were processed using HKL2000 [37] to a resolution of 2.0 Å. The crystals belong to space group P212121 with unit cell dimensions of approximately 68.8 Å × 88.8 Å × 101.8 Å. The Matthews coefficient [38] is approximately 2.4 Å3/Da with a single copy of the LmMetRS catalytic core present in the asymmetric unit. Data collection statistics are presented in Table 1.

Table 1
Data collection statisticsa

The structure was solved by molecular replacement using the BALBES [39] web server, which used the protein A chain of the MetRS:tRNAMet complex from A. aeolicus (Protein Data Bank Identifier (PDBID) 2csx) [40] as the search model. The molecular replacement solution was then fed into ARP/wARP [41] for automated rebuilding of the model. All waters and suspect regions of this auto-traced model were removed and then iterated manual model building/rebuilding with Coot [42] and restrained refinement with REFMAC5 [43] continued.

The protein was cocrystallized with methionine and MgATP and it was clear from the density that the first reaction catalyzed by MetRS had occurred and the product MetAMP was present in the active site. The PRODRG server [44] was used to generate ideal coordinates and refinement restraints for MetAMP, which was then placed into the difference density peaks using Coot. Large difference density peaks still remained in the active site so the product pyrophosphate and a tightly coordinated magnesium ion were modeled into the characteristically-shaped density using Coot. LmMetRS is not a Zn-binding MetRS because it lacks the Zn-binding knuckle motif with the characteristic Zn-coordinating Cys ligands in its CP1, but the final model includes a Zn2+ ion. This ion is located at the surface of the Rossmann fold domain away from CP1 and the active site, and is coordinated by the sidechains of His258 and Glu307 and by two water molecules. It was placed in a difference density peak at this site because of the size of the peak and the roughly tetrahedral coordination geometry, though the distances from the four ligands to the metal are longer than typically seen for a Zn-coordination environment. We thus believe it is an artifact that resulted from the ZnCl2-containing purification buffers and does not have biological significance.

In the final cycles of refinement, perturbational displacement of the protein was described by five translation/libration/screw (TLS) groups as identified by the TLS motion determination (TLSMD) server [45, 46] and TLS parameters were refined for each group prior to restrained refinement in REFMAC5. Model quality was monitored and validated using Coot and MolProbity [47]. The CCP4 suite of programs [48, 49] was used extensively for all steps from data preparation through refinement. Model refinement statistics are presented in Table 2. Molecular figures were created and rendered with PyMOL [50].

Table 2
Model refinement statistics

2.4. Accession Number

Atomic coordinates and structure factors for LmMetRS in complex with MetAMP and Mg-pyrophosphate have been deposited in the Protein Data Bank [51] (http://www.pdb.org) with accession number 3KFL.

3. Results

3.1. The structure of the catalytic core of L. major MetRS

The 2.0 Å crystal structure of the catalytic core of LmMetRS, spanning residues 206-747 of the 747 residue protein, has been determined with one protein molecule in the asymmetric unit with good crystallographic and stereochemical statistics (Tables 1 and and2).2). Residues 207 to 736 are well defined in the electron density map; only the 22 amino acid N-terminal expression tag, residue 206, and the final 11 residues at the C-terminus, Thr737-Gln747, are absent from the structure. The polypeptide adopts an elongated shape containing the class I aaRS core catalytic Rossmann fold domain with a connective peptide insertion comprised of two contiguous parts named CP1 and CP2, the stem-contact-fold (SCF), and the anticodon-binding helix bundle (Fig. 1).

In the LmMetRS crystals, protein molecules are not involved in biologically relevant contacts with any neighbors since the calculated ΔG of the strongest protein-protein interface is only -3.3 kcal/mol-1 according to the PISA server [52]. The lack of evidence for multimers in the crystal structure is in agreement with results from dynamic light scattering and size-exclusion chromatography with the N-terminally truncated protein used in this study; both methods suggest the presence of monomers in solution. Of note, the full-length protein also behaves like a monomer in solution according to these same two biophysical techniques (data not shown).

3.2. The binding of two products and magnesium by L. major MetRS

The protein was crystallized in the presence of the substrates L-methionine and MgATP; however, we find the two products of the amino acid activation reaction, MetAMP and pyrophosphate, with a bridging Mg2+ ion, in well-defined density in the active site (Fig. 2a). These products are in direct contact with residues of the Rossmann fold domain and the SCF, and in water-mediated contact with CP1 (Fig. 1 and Fig. 2). Structural features important for catalysis, derived from previous MetRS structures [40, 53], are all free from contacts with other molecules in the crystal and are engaged in numerous contacts with the two products.

Figure 2
Products bound in active site of L. major methionyl-tRNA synthetase

3.2.1. The pyrophosphate ion

This is the first time that the pyrophosphate product has been observed in the active site of a Class I aaRS. The pyrophosphate ion makes numerous interactions with surrounding groups:

  1. the protein: favorable electrostatic interactions are made by the “first” phosphoryl group of the pyrophosphate (corresponding to the β phosphoryl group of the ATP substrate as deduced from comparison with the structure of the Geobacillus stearothermophilus (this organism was previously known as Bacillus stearothermophilus but we will refer to it as G. stearothermophilus to help propagate the new name) TrpRS:ATP complex [54]) with the imidazole groups of His225 and His228 and the amino groups of Lys522 and Lys525, and by the “second” phosphoryl group (the γ phosphoryl group of the ATP substrate) with the amino group of Lys261 (Fig. 2b). In addition, the sidechains of Asn221 and Ser526 contact the second phosphoryl group of the pyrophosphate. Lys525 is the only sidechain interacting with the bridging oxygen of the pyrophosphate. Clearly, the conserved “HIGH” (residues 225-228 in LmMetRS) and “KISKS” (residues 522-526) motifs of the class I aaRS family are crucial for binding the pyrophosphate ion, providing six out of the seven interacting sidechains.
  2. the methionyladenylate: two oxygen atoms from the first phosphoryl group of the pyrophosphate come within 3.2 Å of the phosphoryl oxygen atoms from MetAMP. This would be an unfavorable interaction if all phosphoryl oxygen atoms would be deprotonated, but the pH of the crystallization medium is ~7.3 so there is at least one hydrogen on the pyrophosphate ion available to mediate the interaction with at least one phosphoryl oxygen atom of MetAMP.
  3. the magnesium ion: one oxygen of the first and one oxygen of the second phosphoryl group from the pyrophosphate are engaged in short contacts with the Mg2+ ion, one of about 2.3 Å and one of about 2.4 Å. These contacts may contribute to the rather eclipsed conformation of the pyrophosphate moiety bound to LmMetRS.
  4. water molecules and ethylene glycol: the first phosphoryl group of the pyrophosphate interacts with one water molecule and the second phosphoryl moiety with two water molecules and a hydroxyl from an ethylene glycol molecule. Ethylene glycol was used to cryoprotect the crystal and it is safe to assume that the interacting hydroxyl group is replaced by a water molecule under physiological conditions. Hence the second phosphoryl group remains much more hydrated upon binding to LmMetRS than the first phosphoryl moiety.

3.2.2. The magnesium ion

The Mg2+ ion has six atoms arranged in a square bipyramidal coordination shell and bridges the pyrophosphate and MetAMP products. It is interacting with two oxygen atoms from two different phosphoryl groups of the pyrophosphate (as described in section 3.2.1), one oxygen from the MetAMP phosphoryl group and three water molecules, though one of these is a bit distant for a typical Mg ligand at approximately 2.5 Å (Fig. 2b). It is of interest that the Mg2+-ion in our complex of L. major MetRS with two products engages oxygen atoms from three different phosphoryl groups: the α-phosphoryl from MetAMP and the two phosphoryl groups of the pyrophosphate. This arrangement is reminiscent to that discussed for the pre-transition state in the TrpRS-catalyzed reaction [55], where the Mg2+-ion interacts with α, β and γ phosphate oxygen atoms of ATP.

One of the water molecule ligands in close contact with the Mg2+ ion also interacts with the carbonyl oxygen of MetAMP and with the sidechain of Asp486. The second water in close contact with Mg2+ does not interact with the protein directly but itself makes a water-mediated contact to the sidechain of Asp336. The slightly distant water ligand makes a hydrogen bond with the carbonyl oxygen of Ser335 and the second phosphoryl group of the pyrophosphate. Hence, while the magnesium ion is interacting directly with the pyrophosphate anion and the MetAMP intermediate, its interactions with the synthetase are all water-mediated. One of the water-mediated contact residues, Asp486, is conserved among the MetRS sequences but Asp336 is not.

3.2.3. The methionyladenylate intermediate

An interesting feature of MetRS is the large conformational change that occurs in the active site upon methionine binding. Indeed, the binding pocket for the amino acid sidechain is not even present in the absence of the amino acid and this induced fit mechanism is a major reason why MetRS is able to discriminate methionine well from most other amino acids [53, 56, 57]. Accordingly, the methionyl moiety of MetAMP is completely buried and places its hydrophobic sidechain in a deep pocket formed by residues Pro216, Ile217, Tyr218, Tyr219, Asp256, Trp443, Ala446, Leu447, Asn449, Tyr450, Ile487, and His491 (Fig. 2c).

The phosphoryl group is interacting with the backbone nitrogen of Tyr219, the amino group of Lys525 from the KISKS motif, and with the pyrophosphate and magnesium ions (as described in sections 3.2.1. and 3.2.2., respectively). The ribose group is contacting residues Ser231, Asp486, and the backbone nitrogen of Gly484. The adenine ring is interacting with the carbonyl oxygen and backbone nitrogen of Trp516, the carbonyl oxygen of Ile523, the sidechain amide of Lys522 from the KISKS motif, and approaches the sidechain of Trp515, though stacking interactions are not observed (Fig. 2d). The equivalent Trp288 in the A. aeolicus MetRS structure [40], however, is present in an alternate rotamer that is involved in a stacking interaction with the adenine base of a MetAMP analog so it is possible that Trp515 does stack with the adenine at some point in the catalytic cycle.

3.3. Comparison of ligand binding and active site conformation in L. major and E. coli MetRS

These include EcMetRS in apo form [58], in complex with methionine [57] and with intermediate product analogs [53], apo Pyrococcus abyssi MetRS [59], apo Thermus thermophilus MetRS [60], the binary complex of Aquifex aeolicus MetRS with tRNAMet and its ternary complex with 5’-O-[N-(L-methionyl)-sulfamoyl] adenosine (MetSA) and tRNAMet [40], and most recently that of Mycobacterium smegmatis MetRS in complex with Met and with both Met and adenosine [61] (Supplementary Table 1). A comparison of these structures (Fig. 3) shows that the active site of the LmMetRS structure is in the most compact conformation observed to date. In particular, the loop within the SCF (colored red in Figure 1), which contains the class I signature KISKS motif, and the CP1 peptide (colored cyan in Figure 1) are in a “closed” conformation and are much closer to the bound ligands than seen before (Figs. 3a and 3b).

Figure 3
L. major MetRS and ligands compared to other methionyl-tRNA synthetases

MetAMP in our ternary complex of LmMetRS shows a very similar binding mode as both L-methioninyl adenylate (Metol-AMP) and MetSA in binary complex with EcMetRS [53] (Fig. 3c). The differences between the true product MetAMP and these two analogs are small, Metol-AMP lacks the carbonyl oxygen atom and MetSA has two atom substitutions; the anhydride oxygen and the phosphorus of MetAMP are nitrogen and sulfur, respectively in MetSA. Since neither the carbonyl oxygen nor the two MetSA-substituted atoms are in direct contact with any protein atom in each structure, and since the conformations of the ligands are so similar overall, it is not likely that these changes are responsible for the structural differences observed in the KISKS loop and CP1 between the two enzymes. Rather, the major conformational differences between the active sites of these enzymes are likely due to the presence of the pyrophosphate and the Mg2+ ions in LmMetRS. In the absence of these additional ligands in the active site, there is nothing to stabilize the KISKS loop or CP1 in the closed conformation observed in the LmMetRS structure.

CP1 is observed in several conformations, or is entirely disordered, in the available MetRS structures so is clearly highly flexible. As mentioned, CP1 of the LmMetRS structure displays a significantly more closed conformation than has been observed in previous MetRS structures and folds over the active site (Figs. 3a and 3b). This region of the enzyme sometimes binds one, as seen in EcMetRS, or two zinc ions, but none are bound in CP1 of the L. major enzyme. This is not surprising because LmMetRS lacks the canonical CxxC Zn-binding motifs. The structure of a region of CP1 in the L. major structure, however, greatly resembles the Zn-binding “knuckle” of CP1 in EcMetRS (PDBID 1pfy) [53]. The two CxxC motifs of the E. coli enzyme (residues 145-148 and 158-161) that coordinate the metal are instead SxxD (residues 333-336) and SxxS (residues 359-362), respectively, in the L. major enzyme (Fig. 3b). For the sake of comparison, we will refer to these as the “pseudo-Zn-binding motifs” in LmMetRS. Upon comparison of the positions of the Zn-binding motifs with that of the corresponding pseudo-Zn-binding motifs (Fig. 3b), there is an obvious large hinge motion originating in the two-stranded antiparallel β-sheet at the base of CP1 (the long, curved, two-stranded β-sheet colored cyan at the left-hand side of Fig. 1), whose strands mark the N- and C-termini of this insertion. Interestingly, TLSMD analysis [46] of the both the LmMetRS structure and an EcMetRS structure (PDBID 1pfy) [53] identifies the majority of CP1 as a single pseudo-rigid group with boundaries near the termini of these same helices. These segmented TLS models are also consistent with a hinge being present [62] at the base of CP1.

In addition to the hinge motion of CP1, there is a twist of approximately 180° in the globular region containing the (pseudo-)Zn-binding motifs. This motion results in the CxxC (SxxD in LmMetRS) that is farthest from the active site in the “open” conformation of E. coli MetRS actually forming a new wall in the active site pocket and making water-mediated contacts with the product pyrophosphate in the “closed” conformation of LmMetRS. The net displacement of the (pseudo-)Zn-binding motifs throughout this bending and twisting motion is not less than 26 Å (Fig. 3b). We have captured a transient closed state of CP1 due to the presence of the Mg-pyrophosphate intermediate. Flexibility in this region of the protein is likely important for the catalytic cycle. CP1 appears to have an important role for substrate/product interactions during the amino acid activation reaction but then must move substantially so that the tRNA acceptor stem can access the active site to be charged with the amino acid (discussed in section 3.5).

3.4. Conformational similarities with adenosine-5′ tetraphosphate binding by TrpRS

Tryptophanyl-tRNA synthetase (TrpRS) is a well-studied member of aaRS subclass Ic [10, 20, 54, 63-70] also containing the HIGH and KMSKS motifs, which both interact with the pyrophosphate in LmMetRS (Fig. 2b). Since LmMetRS is the first class I aaRS structure showing how pyrophosphate interacts with the enzyme, it is of interest to compare our structure with that of G. stearothermophilus TrpRS in complex with the competitive ATP analog adenosine-5′ tetraphosphate (AQP) [66]. This structure has been proposed to mimic a transition state in the amino acid activation mechanism in which the KMSKS loop develops tight interactions with the leaving pyrophosphate, represented by the two terminal phosphoryl groups of AQP [66].

The least squares superposition of AQP in TrpRS onto MetAMP in LmMetRS, using only the adenosines plus the phosphoryl group common to the two ligands, shows that the γ and δ phosphorus atoms of AQP fall within 0.8 Å and 0.3 Å, respectively, of the “first” (the β phosphate of the natural ATP substrate) and “second” (the γ phosphate) phosphorous atoms of the pyrophosphate bound to LmMetRS (Fig. 4a). Interestingly, the KMSKS loop in this G. stearothermophilus TrpRS structure adopts a closed conformation very similar to that of the KISKS loop in the L. major structure. The seven residues interacting with the pyrophosphate in the LmMetRS structure (His225 and His228 from the HIGH motif; Lys522, Lys525 and Ser526 from the KISKS motif; plus Asn221 and Lys261) can now be compared with the residues contacting the γ and δ phosphoryl groups from AQP in G. stearothermophilus TrpRS. These sidechains are only partly conserved in the G. stearothermophilus TrpRS enzyme: Thr15 and Asn 18 from TIGN in G. stearothermophilus TrpRS correspond to the HIGH motif in LmMetRS; Lys192, Lys195 and Ser196 from its KMSKS correspond to L. major’s KISKS motif; Ser11 in G. stearothermophilus TrpRS corresponds to Asn221 in LmMetRS. Although Lys261 in LmMetRS does not structurally align with another residue in G. stearothermophilus TrpRS, it appears to be functionally replaced by Lys111 of TrpRS, which likewise interacts with the γ-phosphate [55].

Figure 4
a. The active site of L. major MetRS in complex with MetAMP and Mg-pyrophosphate compared to that of G. stearothermophilus TrpRS in complex with AQP. L. major MetRS and G. stearothermophilus TrpRS [66] have been structurally aligned by least squares superposition ...

As a result of these differences, two of the five charged residues interacting with the pyrophosphate in LmMetRS have been replaced by neutral residues and one charged contact residue is absent in the case of TrpRS. Nevertheless, six residues of G. stearothermophilus TrpRS interact with the γ and δ phosphoryl groups of AQP in a very similar manner as the six corresponding residues of LmMetRS interact with pyrophosphate (Fig. 4a). The postulated position of the Mg2+ ion in G. stearothermophilus TrpRS, represented by the β phosphorus of AQP [66], falls only 1.0 Å away from the observed position of the Mg2+ ion bound to LmMetRS (e.g. compare Fig. 8 in ref. [66] vs. Fig. 4a in the present paper). The deduction that the Mg2+ ion in TrpRS would not interact with protein atoms [66] is in agreement with the fact that the Mg2+ ion in the LmMetRS structure is only involved in water-mediated interactions with protein sidechains. This AQP-bound structure does indeed appear to mimic the pyrophosphate-bound structure as Retailleau et al. [66] proposed.

3.5. Insight into tRNA-binding from comparison with the A. aeolicus MetRS-tRNA complex

The SCF and anticodon-binding helix bundle domains of MetRS are of major importance for recognizing tRNAMet. Insight into the residues involved in this recognition process in the L. major enzyme can be obtained by comparing our structure with the complexes of A. aeolicus MetRS with its cognate tRNAMet [40] (Fig. 4b). The crucial residues implicated in interacting with the anticodon region of tRNA in the A. aeolicus enzyme are identical in LmMetRS and correspond to Asn580, Arg584, Trp653, Phe730 and Lys732 (green triangles in Fig. 5a).

Figure 5Figure 5Figure 5
Comparison of L. major MetRS sequence with other MetRS sequences

In addition to interacting with the tRNA anticodon region in the helical bundle domain, A. aeolicus MetRS contacts the tRNA acceptor stem region with the CP2. When we superimpose the canonical Rossmann fold of the catalytic core of LmMetRS in complex with its two intermediate products and magnesium onto that of the A. aeolicus enzyme in complex with MetSA and tRNAMet (root mean square deviation (RMSD) of ~0.6 Å for 161 Cα atoms) it is apparent that, at the very least, CP1 and the pyrophosphate-binding KISKS loop of the SCF need to be repositioned to permit access of the tRNA acceptor stem into the active site (Fig. 4b). Indeed, CP1 has not been modeled in either of the A. aeolicus MetRS:tRNAMet structures suggesting that it is quite mobile during certain steps in the catalytic cycle as discussed in Section 3.3. In addition to these implied necessary structural rearrangements, the pyrophosphate and magnesium ions very likely have to vacate the active site to allow the terminal adenosine of the tRNAMet to come into close proximity of the MetAMP intermediate. It is plausible that these entities vacate the active site with a concomitant rearrangement of CP1 and the KISKS loop, which would open up the active site for tRNA access.

4. Discussion

4.1. Possible function of domains outside the catalytic core of L. major MetRS

Trypanosomatid MetRS contains an additional domain N-terminal to the catalytic core whose structure is presented here. Compared to the N-terminal domain of the human cytosolic enzyme, this 210-residue domain has ~19% sequence identity overall, and 28% identity for residues 78-175, compared to the N-terminal domain of the human cytosolic enzyme, including a conserved WLEWE motif of unknown significance (Fig. 5b). In mammals, this extra N-terminal domain mediates association of MetRS into a complex of multiple aaRS and several additional proteins [71-73], and it is possible that it may serve the same function in trypanosomatids. The human mitochondrial MetRS, however, has a very small N-terminal domain that bears no similarity to that of the trypanosomatid or human cytosolic MetRS. Despite this greater similarity to the cytosolic enzyme upstream, the catalytic core of trypanosomal MetRS is more closely related to that of human mitochondrial MetRS than to human cytosolic MetRS (30% vs. 18% identity, respectively). Thus, the trypanosomatid enzyme contains features of both the human mitochondrial and cytosolic homologs, which may plausibly be related to the fact that these parasites encode only a single MetRS in their genomes.

Trypanosomatid MetRS enzymes have no C-terminal domain downstream of the catalytic core and consequently lack the dimerization domain present at the C-terminus of the E. coli enzyme [74]. Therefore, based on sequence alone, full length LmMetRS is expected to be monomeric in solution, as indeed is observed (data not shown). However, due to the sequence similarity of the N-terminal domain with that of mammalian homologs, it is still possible that LmMetRS is part of a multi-synthetase complex in vivo.

4.2. MetRS in complex with its products methionyladenylate and Mg-pyrophosphate

The LmMetRS structure is the first MetRS seen with MetAMP and pyrophosphate bound in the active site and, to the best of our knowledge, the first class I aaRS structure in general with the two products of the amino acid activation step and magnesium bound. MetAMP interacts with the enzyme in a manner similar to that seen for the analogs Metol-AMP and MetSA, with a similar methionine binding pocket that results from an induced fit mechanism [57]. The pyrophosphate interacts with sidechains of seven residues: Asn221, His225, His228, Lys261, Lys522, Lys525, and Ser526. Six of these are contributed by the two sequence motifs characteristic of all class I aaRSs. No fewer than five charged residues interact with the pyrophosphate, which might be a reason why its position could be unambiguously determined (Figs. 2a and 2b). Future work in the aaRS field will clarify how our new structure of LmMetRS, which shows for the first time precisely how the pyrophosphate intermediate product is engaged by mobile elements of the enzyme, fits into the large body of work surrounding the catalytic mechanism and transition state stabilization of MetRS [53, 75-78], TrpRs [54, 55, 66, 70] and TyrRS [79-81].

The elucidated structure of LmMetRS has a very compact arrangement of structural elements near the active site. This “closed” structure is incompatible with the tRNAMet binding mode observed in the A. aquifex MetRS complex structures [40], in particular due to clashes of the aminoacyl acceptor arm of the tRNA with CP1 in the conformation observed in the LmMetRS structure (Fig. 4b). Further, the presence of the pyrophosphate product and Mg2+ would make it difficult for the terminal adenosine to reach the MetAMP intermediate (Fig. 4b). Intriguingly, the distance between the terminal adenosine of the CCA triplet in the tRNAMet (Adenosine 73) and the carbonyl group of the MetSA intermediate analog is ~23 Å in the A. aeolicus MetRS:tRNAMet:MetSA complex [40] (Fig. 4b). This implies that even further structural changes are required, in addition to conformational changes needed for the CP1 insertion and the KISKS loop, so that tRNAMet can be charged by MetRS. Nakanishi, et al. note a 15° shift in the helical bundle domain with respect to the Rossmann fold domain toward the active site between apo MetRS structures and their tRNAMet-bound structures of A. aeolicus MetRS [40]. It is clear that still larger structural changes between the anticodon-binding and catalytic regions are required, or perhaps a major change in tRNA conformation, so that the terminal adenosine reaches the active site. Indeed, some of these motions and coordinated movements have been explored biochemically [75] and, more recently, by molecular dynamics simulations [82, 83] and this structure of LmMetRS bound to all of the products of aminoacyl activation reaction largely supports these findings.

4.3. Opportunities for arriving at selective inhibitors targeting L. major MetRS

Human mitochondrial MetRS has higher sequence identity with the trypanosomal MetRS than the cytosolic homolog. The sequence of human mitochondrial MetRS is only 30% identical to the LmMetRS catalytic core overall, but the active sites are much more highly conserved (Fig. 5a). Four of the 27 residues within 5 Å of the products bound in the LmMetRS structure differ between pathogen and host: LmMetRS Tyr218 is a Phe in human mitochondrial MetRS, Trp515 is a His, and Ile523 is a Met (Figs. 5a and 5c). The carbonyl oxygen of Gly514 in LmMetRS, which is a Ser in the human mitochondrial enzyme, points toward the ligand and the observed phi-psi combination of (-90°, -165°) for this glycine is allowed for serine. The Ser sidechain in the human mitochondrial enzyme would thus point away from the ligand towards a hole between the sidechains of Phe238, Lys310, and Asp383, as deduced from the L. major structure whose corresponding residues are respectively, Arg401, Lys485, and Asp558. Among the “second shell” residues surrounding the “contact residues”, five amino acids differ: Val482 and Val483 in LmMetRS, which are both an Ile in human mitochondrial MetRS, and the amino acid stretch of Ser553-Asn554-Phe555, which is Gly-Val-Pro in the human mitochondrial enzyme. These differences between host and parasite enzymes may seem small but selectivity of the anti-aaRS drug mupirocin that targets prokaryotic IleRS [21] has been attributed to differences in only two residues at the active site [22]. In addition, it is clear that MetRS is a very dynamic enzyme which has to undergo considerable conformational changes during its catalytic cycle [82, 83] (See e.g. Figs. 3b and and4b).4b). Therefore, screening of MetRS against compound libraries could result in the discovery of molecules which lock the enzyme in an inactive conformation and may open a road to anti-parasite drug development and subsequent structure-based drug design.

In the anti-codon binding region, three of the five crucial residues in LmMetRS implicated in interacting with tRNA on the basis of the A. aeolicus structure are conserved in human mitochondrial MetRS: Arg584, Trp653, Phe730 (Fig. 5a). Whether the two changes in this anti-codon binding area, i.e. L. major Asn580 and Lys732 which are, respectively, a Gly and Arg in the human mitochondrial enzyme, are sufficient for arriving at selective inhibitors that target this area remains to be investigated.

4.4. Opportunities for arriving at MetRS leads against sleeping sickness and Chagas disease

The sequence of the catalytic core of LmMetRS, whose structure is described in the current paper, is 89% identical to the enzyme in L. infantum, which is closely related to L. donovani [84], and 97% to that of L. brasiliensis. The sequence identity of the catalytic core of LmMetRS with that of both the T. brucei and the T. cruzi enzymes is 68%, but is 100% when only considering the active sites (Supplementary Fig. 1). This implies that knowledge of the LmMetRS structure will aid the development of therapeutics against other trypanosomatid pathogens as well. In fact, efforts to arrive at leads targeting trypanosomatid MetRS have been initiated and have thus far already resulted in low nM inhibitors of the T. brucei enzyme and of parasite growth, as will described elsewhere (Shibata, et al., submitted).

5. Conclusions

We have solved the X-ray crystal structure of methionyl-tRNA synthetase from the trypanosomatid L. major in complex with the two products of the amino acid activation step, methionyladenylate and Mg-pyrophosphate. The pyrophosphate makes extensive contact with the residues that comprise the Class I aaRS consensus sequence motifs (HIGH and KISKS), highlighting their importance. The pyrophosphate stabilizes the KISKS loop and the CP1 region of the connective peptide in conformations that are much more closed around the active site than seen in previous MetRS structures. This affords a glimpse of yet another structural state of these dynamic enzymes along their catalytic trajectory. The conformation of the active site observed in the LmMetRS structure is not compatible with the tRNA-aminoacylation step, however, because the acceptor stem would be occluded from the reaction center by CP1. CP1 is thus a highly mobile unit that must adopt vastly different conformations as the enzyme fulfills its duty of faithfully charging tRNAMet. In addition to contributing to our understanding of the architecture and ligand-binding properties of MetRS enzymes in general, the LmMetRS structure has highlighted amino acid differences between the active sites of the trypanosomatid enzymes and of the human host. The structural information will aid in the creation of selective drugs targeting the parasite enzyme. Indeed, design of trypanosomatid-specific inhibitors targeting MetRS is currently underway.

Supplementary Material

01

Acknowledgments

We would like to thank Jaclyn Delarosa for assistance with protein characterization, Tracy Arakaki for assistance with crystal screening, and other members of MSGPP for support and fruitful discussions. This work was funded by NIAID awards P01AI067921 (Medical Structural Genomics of Pathogenic Protozoa (MSGPP)), R56AI084004, and RO1AI084004. Portions of this research were carried out at the Stanford Synchrotron Radiation Light Source (SSRL), a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences.

Abbreviations used

aaRS
aminoacyl-tRNA synthetase
AQP
adenosine-5′-tetraphosphate
CP1
connective peptide insertion 1
CP2
connective peptide insertion 2
AspRS
aspartyl-tRNA synthetase
LysRS
lysyl-tRNA synthetase
MetRS
methionyl-tRNA synthetase
EcMetRS
E. coli MetRS
LmMetRS
L. major MetRS
EG
ethylene glycol
MetAMP
L-methionyladenylate
Metol-AMP
L-methioninyl adenylate
MetSA
5′-O-[N-(L-methionyl)-sulfamoyl] adenosine
MSGPP
Medical Structural Genomics of Pathogenic Protozoa
PDB
Protein Data Bank
PDBID
PDB Identifier
RMSD
root mean square deviation
SSRL
Stanford Synchrotron Radiation Light Source
TrpRS
tryptophanyl-tRNA synthetase
SCF
stem-contact-fold
TCEP
tris(2-carboxyethyl)phosphine
TLS
translation/libration/screw
TLSMD
translation/libration/screw motion determination

Footnotes

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