Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the

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Homodimer architecture of QTRT2, the noncatalytic subunit of the eukaryotic tRNA-guanine transglycosylase Christina Behrens, Inna Biela, Stéphanie Petiot-Bécard, Thomas Botzanowski, Sarah Cianférani, Christoph P. Sager, Gerhard Klebe, Andreas Heine, and Klaus Reuter Biochemistry, Just Accepted Manuscript • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Homodimer architecture of QTRT2, the noncatalytic subunit of the eukaryotic tRNA-guanine transglycosylase Christina Behrens§#, Inna Biela§#, Stéphanie Petiot-Bécard†, Thomas Botzanowski†, Sarah Cianférani†, Christoph P. Sager§, Gerhard Klebe§, Andreas Heine§, Klaus Reuter§*

§

Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35032

Marburg, Germany †

Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR

7178, 67000 Strasbourg, France #

CB and IB contributed equally to this work.

*Address correspondence to: Klaus Reuter, Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany, Tel.: +49-6421-28-25845, Fax: +49-642128-28994, email: [email protected]

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Abstract The bacterial enzyme tRNA-guanine transglycosylase (TGT) is involved in the biosynthesis of queuosine, a modified nucleoside present in the anticodon wobble position of tRNAsHis,Tyr,Asp,Asn. Although it forms a stable homodimer endowed with two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time. In contrast, its mammalian counterpart constitutes a heterodimer consisting of a catalytic and a noncatalytic subunit, referred to as QTRT1 and QTRT2, respectively. Both subunits are homologous to the bacterial enzyme, yet only QTRT1 possesses all the residues required for substrate binding and catalysis. In mice, genetic inactivation of the TGT results in the uncontrolled oxidation of tetrahydrobiopterin and, accordingly, to phenylketonuria-like symptoms. Due to this fact and the recent finding that mammalian TGT may be utilised for the treatment of multiple sclerosis, this enzyme is of potential medical relevance rendering detailed knowledge about its biochemistry and structural architecture highly desirable. In the present study, we performed the kinetic characterisation of the murine enzyme, investigated potential quaternary structures of QTRT1 and QTRT2 via noncovalent mass spectrometry, and, finally, determined the crystal structure of the murine noncatalytic TGT subunit, QTRT2. In the crystal, QTRT2 is clearly present as a homodimer with striking similarity to that formed by bacterial TGT. In particular, a cluster of four aromatic residues within the interface of the bacterial TGT, which constitutes a “hot spot” for dimer stability, is present in similar constellation in QTRT2.


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Introduction In the vast majority of bacterial and eukaryotic organisms the genetically encoded guanosine at position 34 (the anticodon wobble position) of tRNAsHis,Tyr,Asp,Asn is replaced by queuosine. Remarkably, this strongly modified nucleoside is based on a 7-deazapurine in place of the customary purine scaffold. In bacteria, the biosynthesis of queuosine commences outside of any tRNA with guanosine 5´-triphosphate being converted to 7-aminomethyl-7-deazaguanine (preQ1) by an intricate anabolic pathway.1-7 In a reaction catalysed by the enzyme tRNA-guanine transglycosylase (TGT), preQ1 is then inserted into position 34 of the above-named tRNAs in exchange with the original guanine. Once preQ1 is incorporated into tRNA, an epoxy-dihydroxy-cyclopentyl moiety deriving from the ribose of S-adenosyl-L-methionine is added to the nitrogen of its exocyclic aminomethyl group.8-10 Finally, in a cobalamin-dependent reaction, the resulting epoxyqueuosine (oQ) is reduced to queuosine.11-15 The bacterial TGT enzyme, which catalyses the irreversible exchange of guanine 34 by preQ1, forms a stable homodimer with the tertiary structure of the monomer representing a (βα)8-barrel. This, however, deviates from the classical motif by a number of extensions and insertions, such as a Zn2+-coordinating subdomain placed between strand β8 and helix α8 (for topology scheme see Figure S1). Although the TGT dimer contains two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time.16 The reaction follows a ping-pong mechanism including a covalent intermediate of the enzyme and its tRNA substrate.17-18

The TGT of Shigella, the causative agent of bacillary dysentery, is important for the full virulence of this pathogen because the ability to translate the mRNA of the virF gene is strongly impaired in Shigella tgt- mutants.19-20 The virF gene encodes a transcriptional activator required for the expression of numerous pathogenicity genes. Hurt et al. demonstrated that in vitro the TGT enzyme is able to recognise the virF-mRNA as a substrate and to replace guanine 421 therein by preQ1.21 If, however, this fact accounts for the reduced translation of this mRNA in the absence of a functional tgt gene, has not been proven so far. Nevertheless, the finding that inactivation of the TGT enzyme ACS Paragon Plus Environment

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strongly reduces the pathogenicity of Shigella prompted us to use it as a target for the development of anti-Shigellosis compounds. Based on the high resolution crystal structure of Zymomonas mobilis TGT22 we have meanwhile constructed inhibitors of this enzyme with low to subnanomolar Kivalues.23

Eukaryotes possess a TGT too, and for mice it was shown that genetic inactivation of this enzyme elicits symptoms resembling those of the hereditary disease phenylketonurea.24 Although the underlying mechanism has not been clarified yet, the lack of a functional TGT leads to the uncontrolled oxidation of the phenylalanine hydroxylase cofactor tetrahydrobiopterin. Consequently, TGT deficient mice are impaired in their ability to produce tyrosine from phenylalanine.24 In light of that, it seems of utmost importance to create compounds which specifically inhibit the bacterial enzyme but not the eukaryotic one. For this purpose, comprehensive functional and structural knowledge of the eukaryotic TGT appears mandatory.

Findings suggesting that the TGT enzyme may be exploited for the treatment of multiple sclerosis (MS) provide further motivation for the detailed investigation of mammalian TGT.25 Based on its insertion into DNA, the chemotherapeutic cytotoxic agent 6-thioguanine (6-TG) exerts strong antiproliferative activity. Remarkably, this activity is also observed in the absence of a functional hypoxanthine-guanine phosphoribosyltransferase (HPRT), an enzyme which is prerequisite for the placement of 6-TG into DNA. Varghese et al. showed that the DNA-independent antiproliferative activity of 6-TG strictly relies on TGT, i.e. on its insertion into RNA.25 In the same study, the authors presented the 7-deazaguanine derivative “NPPDAG”, which is a competent substrate of TGT but not recognised by HPRT. Similar to but even more efficiently than 6-TG, this compound rapidly and completely resolves the symptoms of ongoing disease in the murine MS model “experimental autoimmune encephalomyelitis”. The full remission of illness arises from a TGT-dependent antiproliferative effect of NPPDAG on antigen-specific effector memory T cells and central memory T cells, with naive T cells not being affected.25


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Unlike its bacterial counterpart, the eukaryotic TGT constitutes a heterodimer consisting of a catalytic subunit, QTRT1, and a noncatalytic subunit, QTRT2, both being homologous to the bacterial TGT protomer. Accordingly, both subunits contain the three cysteines and the histidine required for Zn2+ coordination. Yet, only QTRT1 possesses all the residues known to be necessary for binding of the substrate base and for catalysis.26-27 Interestingly, this stands in analogy to a further tRNA modifying enzyme, tRNA m1A58 methyltransferase, which, in bacteria, is a homotetramer (a homodimer of homodimers) being able to bind only two tRNA substrate molecules simultaneously.28 The eukaryotic counterpart of this enzyme is a heterotetramer (a homodimer of heterodimers) comprising two catalytic as well as two noncatalytic subunits, which are both homologous to the bacterial tRNA m1A58 methyltransferase protomer.29

Because, in contrast to bacteria, eukaryotes are not able to synthesise queuosine de novo they have to salvage this compound as a micronutrient from the environment.30 Accordingly, the eukaryotic TGT must be able to directly recognise and insert queuine (the queuosine base), which is not a substrate for bacterial TGT, into tRNA. Homology modelling as well as site-directed mutagenesis provided evidence that the exchange of Val233 in bacterial TGT (Z. mobilis TGT numbering) by glycine in QTRT1 leads to an enlargement of the substrate-binding pocket, which is necessary to accommodate the bulky dihydroxy-cyclopentenyl extension of queuine. In addition, the replacement of Cys158 in bacterial TGT by valine in QTRT1 seems to hamper the (irreversible) insertion of preQ1 into the tRNA of eukaryotes, which are not able to further convert this base to queuine.18,31-32 The mutation of Val233 to glycine and Cys158 to valine is, however, not sufficient to convert the substrate specificity of bacterial TGT to that of the eukaryotic enzyme because a Z. mobilis TGT(Cys158Val/Val233Gly) variant is not able to insert queuine into tRNA.18 Hence, the crystal structure of a eukaryotic TGT seems prerequisite to understand its functioning and substrate specificity.

To deepen our knowledge of the mammalian enzyme we determined Michaelis-Menten parameters of the recombinant murine TGT and investigated potential quaternary structures which its


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subunits form in addition to the functional heterodimer. Former work of Boland et al.26 and Chen et al.27 had indicated the existence of both monomeric and homodimeric QTRT1 and QTRT2. Finally, we determined the crystal structure of QTRT2 revealing that it is able to form a homodimer with striking similarity to that of bacterial TGT.


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Materials and methods Overexpression of murine QTRT1 and QTRT2 genes Murine QTRT1 and QTRT2 genes were chemically synthesised by GeneArt™ (Regensburg, Germany) with codons optimised for expression in Escherichia coli (see Figures S2A and S2B for the complete sequences) and inserted into expression vector pASK-IBA13plus (IBA, Göttingen, Germany). In the resulting plasmids, pASK-IBA13plus-mTGT (for the expression of QTRT1) and pASK-IBA13plusmQv1 (for the expression of QTRT2), the respective target gene is fused at its 5´-end to a sequence encoding an N-terminal Strep-tag®II separated from the start codon by a sequence encoding a thrombin cleavage site (Figure S3). For overexpression of murine QTRT1 and QTRT2 genes, either pASK-IBA13plus-mTGT or pASK-IBA13plus-mQv1 were transformed into E. coli BL21 CodonPlus(DE3)-RIPL (Agilent Technologies, CA, USA). Transformed cells were grown at 37 °C in 4 L of 2 × YT medium33 containing 100 mg·L-1 ampicillin and 34 mg·L-1 chloramphenicol until an OD600 ≈ 0.5 was reached. Subsequently, the bacterial culture was cooled down to 15 °C and gene expression was induced by the addition of anhydrotetracycline to a final concentration of 200 µg·L-1. Incubation was continued at 15 °C for a period of 20 h to 24 h whereupon cells were harvested by centrifugation and frozen at -80 °C.

Purification of recombinant murine QTRT1 and QTRT2 The frozen cell pellet was resuspended in 50 mL of lysis buffer (100 mmol·L-1 TrisHCl pH 8.0, 150 mmol·L-1 NaCl, 1.0 mmol·L-1 EDTA) at room temperature. Cell lysis occurred on ice in a Rosett glass jar by ultrasound for 5 to 8 min using a Branson Sonifier 250 endowed with a flat tip (output control 7; 30% duty cycle). Alternatively, cell disruption was achieved using an EmulsiFlex C5™ high pressure-homogeniser (Avestin Europe GmbH). Afterwards, the cell lysate was centrifuged at 50000 × g at 4 °C for 60 min. The clear supernatant was loaded onto a Strep-Tactin® Superflow® column (IBA, Göttingen, Germany) with a bed volume of 10 mL. The column was washed with 180 mL of lysis


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buffer followed by elution of the target protein with elution buffer (50 mmol·L-1 HEPES pH 7.5, 150 mmol·L-1 NaCl, 1.0 mmol·L-1 EDTA, 2.5 mmol·L-1 desthiobiotin). The mQTRT1 or mQTRT2 containing fractions were combined and concentrated to a volume of 5 mL using a Vivaspin® 20 concentrator (cutoff: 30 kDa; Sartorius Stedim Biotech, Göttingen, Germany). The protein solution was then loaded onto a HiLoad 26/600 Superdex200 prep grade column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) for size exclusion chromatography. Elution occurred with a buffer containing 20 mmol·L-1 HEPES pH 7.5, 1000 mmol·L-1 NaCl and 2.0 mmol·L-1 DTT. All chromatographic steps were carried out at room temperature and 2.0 mL·min-1 flow rate using an ÄKTA FPLC system (GE Healthcare BioSciences AB, Uppsala, Sweden).

The mQTRT1 or mQTRT2 containing fractions of size exclusion chromatography were pooled and concentrated to approximately 3 mg·mL-1 using a Vivaspin® 20 concentrator (cutoff: 30 kDa). Subsequently, the Strep-tag®II was proteolytically cleaved from the target protein using 2 U of biotinylated thrombin per mg target protein for 12 to 15 h at room temperature. Both the biotinylated thrombin and the Strep-tag®II were then removed from the solution using Streptavidin Agarose and Spin Filter columns. Biotinylated thrombin, Streptavidin Agarose and Spin Filter columns are components of the Thrombin Cleavage Capture Kit (Merck Millipore, Darmstadt, Germany) and the procedure was done according to the vendor´s protocol. Compared with the natural QTRT1 from mouse, the additional sequence Gly-Ser remained attached to the N-terminal methionine after thrombin cleavage. Compared with the natural murine QTRT2, the additional sequence Gly-Ser-Arg-Asp-ArgMet remained attached to the N-terminal methionine after thrombin cleavage. Moreover, recombinant QTRT2 contains an additional Gly-Leu dipeptide at its C-terminus (Figure S3; UniProtKB/SwissProt sequence identity code of original murine QTRT1: Q9JMA2; that of original murine QTRT2: B8ZXI1). Using a Vivaspin® concentrator (cutoff: 30 kDa), the virtually pure recombinant target proteins were finally concentrated to approximately 6 mg·mL-1 in the same buffer as used for size exclusion chromatography. The yield achieved by this protocol typically amounted to approximately 4 mg QTRT1 or QTRT2 per litre of bacterial culture. The complete removal of the Strep-tagII® and the ACS Paragon Plus Environment

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integrity and homogeneity of the QTRT2 preparation were verified by means of mass spectrometry (mass spectrometry facility of the Philipps University of Marburg). The measured molecular masses of QTRT1 and QTRT2 amounted to 44234 Da and 47161 Da, respectively, which is in good agreement with the calculated molecular masses of the recombinant proteins after thrombin cleavage (44237 Da and 47161 Da, respectively).

Kinetic characterisation of recombinant murine TGT The determination of Michaelis-Menten parameters for mTGT (mQTRT1/mQTRT2) was done as described by Biela et al.18 monitoring the insertion of [3H]-labelled guanine (7.5% [8-3H]-guanine; 12 Ci·mmol-1, Hartmann Analytic, Braunschweig, Germany), preQ1 (7.0 Ci·mmol-1, Moravek Biochemicals Inc., CA, USA) or queuine (7.0 Ci·mmol-1, Moravek Biochemicals Inc.) into tRNA. QTRT1 and QTRT2 were used at a concentration of 150 nmol·L-1, respectively. The preparation of the required unmodified E. coli tRNATyr (ECY2)34 via in vitro transcription was done using the RiboMAX™ Large Scale RNA Production System-T7 (Promega, Fitchburg, MA, USA) according to the vendor´s protocol. The concentration of tRNA was determined via UV photometry (λ = 280 nm) using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). In order to determine Michaelis-Menten parameters for guanine, preQ1 and queuine (whose concentrations varied between 0.5 µmol·L-1 and 15 µmol·L-1, respectively), the concentration of tRNATyr was kept constant at 15 µmol·L-1. For the determination of Michaelis-Menten parameters for tRNATyr (whose concentration varied between 0.5 µmol·L-1 and 15 µmol·L-1 too), [3H]-labelled guanine was used as the second substrate with its concentration fixed at 10 µmol·L-1.

Noncovalent mass spectrometry experiments Briefly, QTRT1 and QTRT2 samples were buffer exchanged twice against a 0.5 mol·L-1 ammonium acetate pH 8.0 buffer using microcentrifuge gel-filtration columns (Zeba 0.5 mL, Thermo Scientific). Next, protein concentration was determined by measuring absorbance at 280 nm (Nano-Drop 2000 spectrophotomoter, Thermo Scientific). Using microcentrifuge gel-filtration columns (NAP-5, GE ACS Paragon Plus Environment

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Healthcare), tRNATyr was desalted three times, ammonium acetate buffer concentration was set to 0.25 mmol·L-1, pH 7.5 and tRNATyr concentration was determined by measuring absorbance at 260 nm. Noncovalent mass spectrometry experiments were performed on a Q-TOF mass spectrometer (Synapt G2 HDMS, Waters) connected to an automated chip-based nanoESI source (Triversa Nanomate, Advion Biosciences) operating in the positive ion mode. The mass spectrometer was externally calibrated before experiments with singly charged caesium iodide clusters diluted to 2 g·L-1 in a 1:1 (v/v) water:isopropanol solution. In order to preserve noncovalent QTRT dimers and complexes with tRNATyr as well as to improve ion desolvation and ion transfer, tuning parameters were carefully optimised as described below, Vc=140 V, Pi= 6 mbar. Native MS data interpretation was performed using MassLynx v4.1 (Waters).

Crystallisation of mQTRT2 The protein solution used for crystallisation purposes contained mQTRT2 at a concentration of 6 mg·mL-1 dissolved in 20 mmol·L-1 HEPES pH 7.5, 1000 mmol·L-1 NaCl and 2 mmol·L-1 DTT. Prior to crystallisation, the solution was centrifuged at 16000 × g at 4 °C for 60 min in order to precipitate higher aggregates of the protein. With the monodisperse supernatant (as shown by dynamic light scattering using a Spectro Size 300 - Nano Biochem Technology, Lüneburg, Germany), crystallisation experiments were carried out at 18°C using vapour diffusion technique. An initial search for crystallisation conditions using commercially available crystallisation screens was done by the protein crystallisation facility of the Philipps University of Marburg, MARXTAL. Rod-shaped crystals were obtained under condition 72 of the PACT Suite (200 mmol·L-1 sodium malonate, 100 mmol·L-1 Bis-Tris propane pH 6.5, 20% (w/v) PEG 3350) (Qiagen, Hilden, Germany). For optimisation of this crystallisation condition, 24 well-Chryschem Plates (hanging drop) as well as the Additive Screen from Hampton Research (Aliso Viejo, CA, USA) were used. While varying pH, sodium malonate concentration and PEG3350 amount did not lead to any improvement, the additive betaine hydrochloride turned out favourable to crystal growth and diffraction behaviour. ACS Paragon Plus Environment

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The optimised reservoir solution contained 200 mmol·L-1 sodium malonate, 100 mmol·L-1 Bis-Tris propane pH 6.5, 25% (w/v) PEG 3350, and 10 mmol·L-1 betaine hydrochloride. For the production of large crystals used for diffraction experiments, 2.5 µL of protein solution were mixed with 2.5 µL of optimised reservoir solution whose volume amounted to 1.0 mL.

Data collection, structure determination, model building and refinement Prior to data collection, mQTRT2 crystals were transferred to reservoir solutions containing increasing proportions (8%, 12%, 16%, and, finally, 20% (v/v)) of PEG 400, which was used as cryoprotectant. Eventually, crystals were vitrified in liquid nitrogen for data collection at 100 K. Data for structure determination and refinement were collected at the Helmholtz-Zentrum Berlin, BESSY II MX-beamline BL14.135 equipped with a Pilatus 6M detector (DECTRIS Ltd., Baden-Daettwil, Switzerland) at a wavelength of 1.282730 Å (absorption maximum of Zn2+; data set used for structure determination via SAD) or 0.918400 Å (data set used for refinement). Subsequently, both data sets (collected from the same crystal) were processed using the programme XDS36. The crystal belongs to space group P21212, which was verified by XPREP (Software for Data Preparation & Reciprocal Space Exploration version 2013/1, Bruker AXS, Madison, WI). It contains two mQTRT2 polypeptide chains within the asymmetric unit and exhibits a solvent content of 49.2% resulting in a Matthews coefficient of 2.4 Å3·Da-1. Details of data collection and refinement statistics are summarised in Table 1.


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Table 1. Crystallographic data collection, processing and refinement statistics PDB ID

6FV5

Dataset used for

SAD

Refinement

Data collection and processing Wavelength (Å)

1.282730

0.918400

Space group

P21212

P21212

a = 62.0, b =118.7, c = 124.8

a = 61.9, b = 118.5, c = 124.6

50 – 2.30

50 – 2.18

1089882 (171499)

303570 (33494)

78969 (12669)

46914 (6374)

Unit cell (Å) a

Resolution range (Å) Total No. of reflectionsa No. of unique reflections

a

a

Completeness (%)

99.4 (99.0)

96.4 (82.2)

a

13.8 (13.5)

6.5 (5.3)

a

21.9 (4.4)

15.6 (2.7)

8.3 (55.6)

7.9 (53.5)

48.3

35.3

Multiplicity

Mean I/σ(I) Rsym

a,b

(%) 2

Overall B factor from Wilson Plot (Å )

Refinement statistics Resolution range in refinement (Å) c

24.78 – 2.18

d

Rwork /Rfree (%)

16.7/20.4

No. proteins per asymmetric unit

2

No. protein residues

377/378

2+

No. Zn ions

2

No. water molecules e

209

2

Mean B factor (Å ) protein/water/Zn2+/other ligands

41.8/41.4/32.5/56.5

rmsd bond length (Å)

0.008

rmsd bond angles (°)

0.960

f

98.1

f

Ramachandran allowed (%)

1.9

Ramachandranf outliers (%)

0

Ramachandran favoured (%)

a

Values in brackets are statistics for the highest resolution shell

b

=

∑ − ∑

c

=

, with I representing the observed intensity and Ī representing the average intensities for multiple measurements.

∑ | − | ∑ | |

d

Rfree was calculated as Rwork but on 5% of the data excluded from the refinement.

e

calculated by MOLEMAN

f

calculated by MolProbity38

37


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The positions of the two Zn2+ ions were determined by SHELXD39 from the HKL2MAP package (version 0.2)40 with the correlation coefficient for the best solution amounting to 39. The calculation of initial phases from the Zn2+ positions as well as subsequent autotracing was done via the programme SHELXE.41 Autotracing generated 624 amino acid residues within 24 peptide chains, which were assigned to two macromolecules (chain A and B). The completeness of the model was improved by means of ARP/wARP (version 5.0)42 implemented in the CCP4 programme package (version 6.4.0).43-44 The target number of residues was 846 with a solvent content of 49.2%. Thereof, 705 residues within five polypeptide chains were built within 190 cycles. Subsequently, the two Zn2+ ions as well as water molecules were added manually using Coot.45 The dataset collected at a wavelength of 0.918400 Å was used for the refinement of the model by means of the programme Phenix (version 1.10.1-2155)46 with intermittent cycles of model building in Coot. TLS refinement involving the partitioning of the model into 14 TLS groups defined by the programme as well as noncrystallographic symmetry restraints were applied in every cycle of Phenix. The B values for all atoms were refined isotropically. After several cycles of refinement, the increased quality of electron density clearly revealed two alternative side chain conformations for Glu372 (chain A) and for Cys392 (chains A and B) as well as the presence of one PEG and one malonate molecule. The refinement of the occupancies of the named side chains and that of the PEG molecule was done during following cycles. In addition, another 50 residues were built resulting in 377 residues within macromolecule A and 378 residues within macromolecule B. Accordingly, the completeness of the final model amounts to 89.2%. Finally, the weighting of the X-ray data between stereochemistry and atomic displacement parameters were optimised by Phenix leading to the refinement statistics summarised in Table 1.

Homology modelling of QTRT1 A template search using SWISS-MODEL and the amino acid sequence of murine QTRT1 revealed nine possible templates sharing a sequence identity between 40.63% and 43.73%. Based on these templates, homology models were build according to the workflow of SWISS-MODEL.47-48 The


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final model, based on the template provided by pdb entry 2ASH, was selected by means of visual inspection and the QMEAN4 criteria.49 After replacing chain B in the crystal structure of QTRT2 with the chosen homology model of QTRT1, a thorough minimisation using the Yeti force field with a convergence criterion of 0.01 kcal·mol-1 was done,50-51 followed by a 100 ns molecular dynamic simulation using Desmond and the OPLS2005 force field.52 The protein-protein interfaces were analysed using DrugScore-PPI.53

Figure preparation Structural figures were prepared using Pymol (http://www.pymol.org).

Protein data bank accession code Coordinates and structure factors have been deposited under accession code 6FV5.


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Results and discussion Kinetic characterisation of recombinant murine TGT In 2009, Boland et al. reported the recombinant production of murine QTRT1 and QTRT2 in Escherichia coli.26 Although the recombinant QTRT1/QTRT2 heterodimer was proven to be enzymatically active when bulk tRNA from yeast (which naturally lacks queuosine) was used as substrate, no Michaelis-Menten parameters were determined for the murine TGT. Kinetic analysis was reported later by Chen et al. only for the human enzyme.27,32 In the present study, we quantified Km as well as kcat values of the recombinant murine TGT, whose subunits were produced separately and combined at a molar ratio of 1:1 for kinetic measurements. Unmodified tRNATyr from E. coli served as tRNA substrate and the insertion of 3H-labelled guanine, preQ1 or queuine into it was used to monitor enzyme activity.

Depending on the substrate base, the kcat values for the murine enzyme were determined to be between 1.7 · 10-3 s-1 and 7.5 · 10-3 s-1 (Table 2, for progress curves and resulting Michaelis-Menten plots see Figure S4) and, accordingly, are comparable to those published for the human enzyme.27,32 As shown for the human TGT, Km(guanine) and Km(tRNATyr) of the murine TGT are virtually identical (1.8 µmol·L-1 and 2.0 µmol·L-1, respectively), although these values are somewhat higher than the corresponding ones published for the human TGT (0.41 µmol·L-1 and 0.34 µmol·L-1, respectively). Km(queuine) measured for the murine enzyme (0.6 µmol·L-1) is very similar to that measured for the human TGT (0.26 µmol·L-1). In the case of the murine enzyme, we were, however, not able to reproduce the high Km(preQ1) of > 100 µmol·L-1 reported for the human enzyme. Yet, Km(preQ1) as measured for the murine TGT (5.1 µmol·L-1) constitutes the highest Km of the three investigated substrate bases and differs from Km(queuine) by about one order of magnitude. Thus, the murine TGT also reveals a significantly higher affinity to queuine compared with preQ1 clearly favouring the insertion of queuine into tRNA. In line with the results of Boland et al.26 and Chen et al.27 we did not detect any


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measurable enzyme activity for QTRT1 and QTRT2 in the absence of the respective dimer mate (data not shown). Table 2. Kinetic parameters of murine TGT

tRNATyr *

guanine

preQ1

queuine

Km [µmol·L-1]

2.0 ± 0.4

1.8 ± 0.7

5.1 ± 1.7

0.6 ± 0.0

kcat [10-3 · s-1]

7.5 ± 0.2

4.3 ± 1.7

1.7 ± 0.6

3.0 ± 0.1

* using 3H-guanine as substrate base

Potential quaternary structures formed by murine TGT subunits Via an immunoassay performed with the lysates of transiently transfected COS-7 cells producing HA and Myc epitope-labelled murine TGT subunits Boland et al. obtained evidence that QTRT1 and QTRT2 strongly interact to form the functional heterodimeric enzyme.26 In addition, they noticed that the noncatalytic QTRT2 subunit of murine TGT partially self-associates to a homodimer while the catalytic QTRT1 subunit does not. Shortly thereafter, Chen et al. investigated the quaternary structure of recombinant human TGT by means of intact mass analyses.27 They showed that the main portion of an equimolar mixture of QTRT1 and QTRT2 assembles to the functional QTRT1/QTRT2 heterodimer. In addition, however, substantial amounts of both subunits in such a mixture remain monomeric or form homodimers.

In the present study, we carried out mass spectrometry experiments under nondenaturing conditions54 (noncovalent MS) to reinvestigate potential quaternary structures that murine TGT subunits are able to form. First, noncovalent MS analysis of QTRT1 in the absence of QTRT2 revealed that the catalytic subunit is mostly monomeric with a measured mass of 44301 ± 1 Da (Figure 1A, upper panel), in agreement with that calculated for a monomer containing one structural Zn2+ ion (44302 Da). Yet, in line with the finding of Chen et al.,27 a minor peak with a mass of 88612 ± 5 Da corresponding to the homodimeric QTRT1 is also observed (theoretical mass of the QTRT1 dimer including one Zn2+ ion per subunit: 88604 Da) (Figure 1A, upper panel). The ratio of monomeric and


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dimeric QTRT1 (estimated to be 80% and 20%, respectively) does not significantly change at a higher QTRT1 concentration (up to 15 µmol·L-1) (Figure S5A).

QTRT2 alone was next analysed in similar experimental and instrumental conditions as QTRT1. Conversely to the catalytic subunit, noncovalent MS analysis of QTRT2 revealed that the protein mostly exists as a homodimer of 94461 ± 1 Da (compared to a theoretical mass of the QTRT2 dimer plus one Zn2+ ion per subunit of 94452 Da), while a minor ion series corresponds to monomeric QTRT2 (47226 ± 2 Da versus theoretical mass of QTRT2 plus one Zn2+ ion of 47226 Da) (Figure 1A, lower panel). Again, increased QTRT2 concentrations do not affect the ratio of monomeric and dimeric QTRT2 (20% and 80%, respectively) (Figure S5B).

Figure 1: Native mass spectrometry analysis of (A) QTRT1 (upper trace) and QTRT2 (lower trace) (c = 5.0 µmol·L-1, each); (B) -1

QTRT1/QTRT2 mixtures (c = 2.5 µmol·L , each) at different time points; (C) QTRT1/QTRT2/tRNA tRNA

Tyr

Tyr

mixtures at increasing

concentrations. Full scan mass spectra are presented on the left; zoom on the m/z 3375 to 3410 region of mono-

meric QTRT1 and QTRT2 species; zoom on the m/z 4500 to 4580 region of dimeric species.

To monitor the kinetics of QTRT1/QTRT2 heterodimer formation we next analysed an equimolar mixture of QTRT1 and QTRT2 monomers by noncovalent MS at different time points (< 1 min as well as after 3 min and 5 min incubation). After < 1 min incubation, a significant amount of the ACS Paragon Plus Environment

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QTRT1/QTRT2 heterodimer (91528 ± 14 Da compared to a theoretical mass of the heterodimer plus one Zn2+ ion per subunit of 91528 Da) is present in addition to the QTRT1 monomer and the QTRT2 homodimer (Figure 1B). With increasing incubation time, the proportion of the QTRT1/QTRT2 heterodimer increases while the amount of the QTRT1 monomer and the QTRT2 homodimer drops. After 5 min, the large majority of the complex is present as a heterodimer, although, in accordance with the results of Chen et al.27, very minor amounts of QTRT1 monomer, QTRT2 monomer and QTRT2 homodimer are still detected (Figure 1B).

Finally, we investigated the stoichiometry of the complex formed between the QTRT1/QTRT2 heterodimer and a tRNATyr substrate (Figure 1C). Noncovalent MS analysis of the tRNA preparation alone revealed substantial heterogeneity with the detection of several entities of 27 kDa, that could be undoubtedly assigned to tRNA species differing in the degree of phosphorylation as well as in the presence or absence of particular nucleotides at the 5´ or 3´ end (Figure S6). Such heterogeneities are classically observed in tRNA analysis and do not affect the recognition of tRNATyr by the TGT enzyme.17,34,55 We next performed titration experiments involving a fixed concentration of QTRT1/QTRT2 dimer and increasing amounts of tRNATyr (Figure 1C). Noncovalent MS analysis of 1:1:1 molar ratio of QTRT1, QTRT2 and tRNATyr reveals that the most abundant ion series corresponds to a 1:1:1 QTRT1:QTRT2:tRNATyr complex (119469 ± 15 Da). Less intense ion series corresponding to monomeric QTRT1, monomeric and homodimeric QTRT2 and QTRT1/QTRT2 heterodimer without bound tRNATyr are also detected. The presence of an excess of tRNATyr results in a strong increase of QTRT1:QTRT2:tRNATyr signals concomitant with a decrease of RNA-free proteins, indicating a stabilising effect of the tRNA substrate on the QTRT1/QTRT2 heterodimeric complex. Even at a five-fold molar excess of tRNATyr, no ternary complex containing more than one tRNATyr molecule is detectable (Figure 1C), in favour of a specific 1:1:1 QTRT1:QTRT2:tRNATyr complex. Not surprisingly, this proves that, in analogy to the bacterial enzyme, the functional eukaryotic TGT is able to bind only one tRNA substrate at a time.


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Structure determination of QTRT2 and overall structure of the monomer As a prerequisite for obtaining information about the three-dimensional architecture of eukaryotic TGT, we performed crystallisation trials with both recombinant murine TGT subunits. While, up to now, QTRT1 as well as the functional QTRT1/QTRT2 complex have appeared recalcitrant to crystallisation we were able to obtain diffraction quality crystals of QTRT2. Crystallisation was performed as described in the Materials and Methods section followed by structure determination via SAD using the naturally bound Zn2+ ion as the anomalous scatterer. The structure was refined at a resolution of 2.18 Å to an R-factor of 16.7% and an Rfree of 20.4% (data collection and refinement statistics are summarised in Table 1). The QTRT2 crystals belong to space group P21212 with the asymmetric unit containing two polypeptide chains, each coordinating one Zn2+ ion. Regarding chain A of the final model, no density can be allocated to the six N-terminal residues of the recombinant protein used for crystallisation. These residues, however, constitute an artefact resulting from the cloning procedure (see Figure S3) so that, with respect to the natural QTRT2 as present in the mouse, no N-terminal amino acid is missing. In the numbering system used in the structural model, the first residue of chain A corresponds to Met1, in conformance with the numbering of the UniProtKB sequence entry of murine QTRT2 (accession code: B8ZXI1). Regarding chain B, two of the artificial amino acids preceding the N-terminal methionine of the natural murine QTRT2 are visible in the structure and referred to as Arg-1 and Met0, respectively. In both polypeptide chains, an expanded segment comprising Cys292 to Gln326 obviously is disordered, as no corresponding electron density is visible in the map. Hence, these residues are omitted from the structural model. Furthermore, in both chains the two C-terminal residues (GlyLeu) of the recombinant protein are invisible in the electron density map and, therefore, not included in the model. Similar to the six N-terminal residues, these residues emerged from the cloning procedure and do not exist in the natural protein. Accordingly, the C-terminal residue of both chain A and B in the structural model is Phe415, which constitutes the C-terminal residue of QTRT2 as present in


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the mouse. Ultimately, no electron density can be assigned to a short stretch comprising Gly21 to Ala23 of chain A, which is why this section is missing in the final model. In addition to 209 water molecules, the structure contains a fragment of a polyethylene glycol molecule as well as one malonate, both being components of the reservoir solution used for crystallisation (Figure S7).

The overall fold and topology of QTRT2 strongly resembles that of the bacterial TGT monomer (Figure 2). Accordingly, the bound Zn2+ ion differs neither in position nor the way it is coordinated from that in the bacterial enzyme (Figure S8). Yet, there are some conspicuous differences within the area corresponding to the substrate-binding pocket and the active centre of bacterial TGT. Nearly no amino acids which accomplish the binding of the substrate base and catalysis in bacterial TGT are conserved in QTRT2. Accordingly, the catalytic nucleophile, Asp280 (Z. mobilis TGT numbering), is replaced by Glu272 in QTRT2. In Z. mobilis TGT, the substrate base (preQ1 or guanine) is sandwiched between the side chains of Met260 and Tyr106 and forms hydrogen bonds (H-bonds) to the side chains of Asp102, Asp156 and Gln203 as well as to the main chain amide of Gly230 (Figure 2C). Of these residues, only Gly230 is conserved in QTRT2 (Gly221) (Figure 2D; for structure based sequence alignment see Figure S9). While both Asp102 and Asp156 are replaced by serines (Ser95 and Ser145, respectively), the position of Gln203 is occupied by a glutamate (Glu194) and Met260 corresponds to a cysteine (Cys252) in QTRT2. In Z. mobilis TGT, Tyr106 (replaced by phenylalanine in most other bacterial TGT enzymes) is located within helix αA (Gly104 to Leu111). In QTRT2, however, helix αA does not exist but is replaced by a loop region (His97 to Pro104) blocking a cavity, which, in bacterial TGT, accommodates ribose 34 and phosphate 35 of the substrate tRNA (Figures 2, S10A, S10D and S10E; for topology scheme see Figure S1).


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Figure 2: Ribbon representations of the subunit structures of Z. mobilis TGT and murine QTRT2. β-strands and α-helices 2+

forming the (βα)8 barrel scaffold are coloured blue and red, respectively. The Zn -binding subdomain inserted between β2+

strand 8 and α-helix 8 is shown in green, the Zn ion as a bright blue sphere. Extensions and further insertions into the (βα)8 barrel are coloured grey. Both N- and C-terminus are indicated. (A) Z. mobilis TGT; the bound preQ1 molecule indicating the position of the active site is shown in stick representation (carbon atoms in yellow, nitrogen atoms in blue and oxygen atom in red). (B) QTRT2; the visible borders of loop βGβH are indicated by red circles. (C) Detail of the Z. mobilis TGT 56

showing the bound preQ1 base (pdb-code: 1P0E). Residues involved in substrate base binding, the active site nucleophile, Asp280, and the N-terminal part of loop β6α6 are in stick representation (carbon atoms of preQ1 in yellow, those of TGT in grey, nitrogen atoms in blue and oxygen atoms in red). In the preQ1-bound state, the carbonyl oxygen of the Leu231/Ala232 peptide bond is oriented to the substrate-binding pocket and forms an H-bond to the exocyclic amino group of preQ1. Hbonds are depicted as dashed yellow lines. (D) Detail of QTRT2 corresponding to that of Z. mobilis TGT shown in (C). Residues corresponding to those interacting with the substrate base in Z. mobilis TGT as well as the N-terminal part of loop β6α6 are shown in stick representation (colour coding as in (C)), H-bonds as dashed yellow lines.

Furthermore, in bacterial TGT the loop connecting strand β6 and helix α6 contains a peptide bond (in Z. mobilis TGT formed by Leu231 and Ala232) which plays an important role in the recognition of the substrate base. Depending on whether guanine or preQ1 is bound, this peptide bond has


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to acquire a different geometry, i.e. the exchange of guanine by preQ1 during catalysis is concomitant with a flip of this bond. Both conformations are stabilised from the protein interior by the side chain of Glu235, which changes its protonation state in the course of the Leu231/Ala232 peptide flip.57 In QTRT2, such interplay between a peptide bond and a nearby glutamate is not possible. Not only is Glu235 replaced by a proline (Pro226) in QTRT2 but also takes the β6α6 loop a significantly different course compared with that of the bacterial TGT precluding the interactions mentioned above (Figures 2C and 2D). Leu231 and Ala232 performing the peptide flip upon substrate base exchange in Z. mobilis TGT are replaced by Phe222 and Gln223 in QTRT2. Of note, the side chain oxygen of Gln223 forms H-bonds to its own main chain amide as well as to the main chain amides of Gly221 and Phe222. In doing so, it renders the binding of the preQ1 base sterically impossible because its side chain would clash with the preQ1 exocyclic aminomethyl group. Also Asp220 (replacing Gly229 of Z. mobilis TGT) prevents preQ1 from binding as its side chain would clash with the exocyclic carbonyl oxygen of a hypothetically bound preQ1. The Asp220 side chain is locked into position by H-bonds formed with the sulfhydryl moiety of Cys252 and the carboxyl group of Glu194 (Figures 2C and 2D) with the latter interaction clearly indicating the protonation of one of the involved carboxyl moieties. In summary, the described observations undoubtedly show that QTRT2 is neither able to bind any substrate base nor to bind the anticodon loop of a tRNA, in line with the fact that it constitutes the noncatalytic subunit of the eukaryotic TGT heterodimer.

A further apparent structural difference between the bacterial TGT protomer and QTRT2 is present within the Zn2+-binding subdomain. A short turn (Thr295 to Gly298) connecting strands βG and βH in bacterial TGT is replaced by an extended putative loop comprising 45 residues (Thr287 to Phe331) in QTRT2. Of these, however, only Thr287 to Asp291 as well as Glu327 to Phe331 are visible in the electron density map while the major part of this presumptive loop is obviously disordered or scattered over multiple conformations. As the Zn2+-binding subdomain is deemed important for the correct orientation of the substrate tRNA during catalysis it is tempting to speculate that loop βGβH in QTRT2 may further support this function and becomes ordered upon tRNA binding. Multiple seACS Paragon Plus Environment

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quence alignment reveals that this loop is obviously present in all eukaryotic QTRT2 subunits although it varies considerably in sequence and length among different organisms. In contrast, it is not detectable in the primary structure of any QTRT1 subunit, where, as in bacterial TGT, a short turn connects strands βG and βH (Figure S9).

Quaternary structure of QTRT2 and comparison with that of bacterial TGTs The two QTRT2 polypeptide chains present in the asymmetric unit unambiguously form a homodimer with striking similarity to the bacterial TGT dimer (Figure 3). As calculated by the programme PISA,58 the contact area between both subunits amounts to 1682 Å2, which is virtually identical in size to that of the Z. mobilis TGT dimer interface (ca. 1660 Å2).59 As in the bacterial TGT, the dimer interface is mainly established by residues located in or close to helices α1, αE, αF and α2c.

In former publications, we presented evidence that in Z. mobilis TGT a cluster of four aromatic amino acids, namely Trp326, Tyr330, His333 (all within or next to helix αE), and Phe92´ (helix α2c´of the dimer mate), provides a particularly large contribution to dimer stability (Figure 4A).59-60 As the subunits are related to each other by a two-fold axis of symmetry, the mentioned “hot spot” is found twice in the interface. In the TGT of Thermotoga maritima (in addition to Z. mobilis TGT the only bacterial TGT of known structure so far; pdb-code: 2ASH), Tyr330, His333, and Phe92´ are conserved (Tyr311, His314 and Phe79´, respectively), while Trp326 is replaced by a phenylalanine (Phe307). However, the reduced size of the monocyclic phenyl ring in comparison with the fused bicyclic indole ring system is compensated by the expansion of the aromatic cluster by the side chain of Tyr302 (helix αD). The latter is locked into position by an H-bond formed to the main chain amide of Arg44´ (Figure 4B), which countervails the lost H-bond formed by the Trp326 side chain NH-group and the main chain carbonyl of Met93´ in Z. mobilis TGT (Figure 4A). Remarkably, a similar arrangement is found in the interface of the QTRT2 homodimer, in which Tyr330, His333, and Phe92´ of Z. mobilis TGT correspond to Tyr363, His366, and Phe84´, respectively. Trp326 of Z. mobilis TGT, however, is replaced by a histidine (His359), whose side chain also provides only a monocyclic aromatic ACS Paragon Plus Environment

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ring system. Here, Tyr354, equivalent to Tyr302 in T. maritima TGT, extends the aromatic cluster by a phenol group, which forms an H-bond to the main chain amide of Gln48´ (Figure 4C). The position of Tyr302 in T. maritima TGT or Tyr354 in QTRT2 is taken by an alanine (Ala321) in Z. mobilis TGT.

2+

Figure 3: Ribbon representations of the Z. mobilis TGT and murine QTRT2 homodimers. Zn ions are shown as bright blue spheres. (A) Z. mobilis TGT homodimer; the subunits are coloured bright green and bright blue, respectively. Darker shades 2+

of colour indicate the Zn -binding subdomains. (B) QTRT2 homodimer; the subunits are coloured bright yellow and bright 2+

grey, respectively, with darker shades of colour indicating the Zn -binding subdomains. Red ellipses indicate the visible borders of loop βGβH.

In QTRT2, the aromatic cluster within the dimer interface is, compared with bacterial TGTs of known structure, expanded by still another two residues, namely His47´ (N-terminus of helix α1´) and His70´ (preceding helix α2A´). Via its side chain, His47´ forms an H-bond to the main chain carbonyl of Ile85´ as well as a hydrophobic interaction with the side chain imidazole of His359. The imidazole of


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His70´ makes a Van-der-Waals contact to His366. In Z. mobilis and T. maritima TGT, both His47´ and His70´ are replaced by prolines (Figure 4).

Figure 4: Aromatic “hot spot” within the dimer interface of (A) Z. mobilis TGT, (B) T. maritima TGT, and (C) murine QTRT2. Colour coding corresponds to that of Figure 3. While the course of the main chain is predominantly shown in ribbon representation, the side chains of residues relevant for the organisation of the aromatic cluster are shown in stick representation. In addition, Met93´ (Z. mobilis TGT), Met80´ (T. maritima TGT) and Ile85´ (QTRT2) as well as the part of the loop-helix motif (loop β1´α1´ plus helix α1´) crossing the respective detail are shown in stick representation (carbon atoms in the same colour as the corresponding ribbon, nitrogen atoms in blue, oxygen atoms in red and sulphur atoms in yellow). Dashed blue lines indicate H-bonds.

In the course of a former project, molecular dynamics simulations provided evidence that, in Z. mobilis TGT, the H-bond formed between the side chain NH of Trp326 and the main chain carbonyl ACS Paragon Plus Environment

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of Met93´ (C-terminus of helix α2c´) is exceedingly persistent and, accordingly, may be important for dimer stabilisation (Figure 4A).59 As in T. maritima TGT, however, the mentioned tryptophan is replaced by phenylalanine, such an H-bond is not able to form in this orthologue (Figure 4B). In QTRT2, Trp326 and Met93´ are exchanged for His359 and Ile85´, respectively. Here too, no H-bond is formed between the His359 imidazole and the main chain carbonyl of Ile85´. The orientation of the imidazole with respect to the Ile85´ carbonyl oxygen as well as an H-bond formed to a well-defined water molecule indicate a conformation of the His359 side chain precluding an H-bond analogous to that observed in Z. mobilis TGT. Similar to the corresponding methionine in Z. mobilis and T. maritima TGT, Ile85´ forms a hydrophobic interaction with Tyr363 (Figure 4C). In Z. mobilis TGT, loop β1α1 (Thr47 to Lys55) and helix α1 (Pro56 to Thr62) play an important role in the stability of the dimer interface as they obviously protect the aforementioned aromatic cluster from water access. This loop-helix motif (i.e. residues 47 to 62) reveals notable flexibility and is believed to adopt a defined conformation only within the intact homodimer while being disordered in the monomeric state. In the course of former projects, we identified a number of TGT ligands and mutations able to force this motif into different conformations concomitant with its (partial) collapse and measurable dimer destabilisation16,61-63 (Figures S10A, S10B and S10C). H-bonds formed by the side chains of His333 and Tyr330 to the main chain carbonyls of Ala48´ and Ala49´, which are located within loop β1´α1´ of the loop-helix motif (Figure 4A), significantly contribute to dimer interface integrity.59 Consequently, the mutation of Tyr330 to phenylalanine precluding the Hbond of this aromatic residue to Ala49´ leads to decreased dimer stability and enzymatic activity.16 Because in QTRT2 the orientation of helix αE towards the loop-helix motif differs somewhat from that observed in the bacterial enzyme, His366 and Tyr363 are not able to form equivalent H-bonds to Thr39´ and Gly40´, which correspond to Ala48´ and Ala49´ of Z. mobilis TGT. Instead, the side chain hydroxyl of Tyr363 forms a strong H-bond (2.5 Å) to the side chain hydroxyl of Ser41´ (Figures 4C and S10D). Notably, the Ser41´ hydroxyl group forms a further H-bond, namely to a well-defined water molecule (Figure 4C). This indicates that the altered orientation of loop β1´α1´ and helix αE in QTRT2 ACS Paragon Plus Environment

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leads, compared with bacterial TGT, to a less pronounced protection of the aromatic cluster from water access. Due to lack of space, no water is present at a similar position in the homodimer interface of wild type Z. mobilis or T. maritima TGT. This may provide one explanation for the difference in stability of the QTRT2 and the bacterial TGT homodimer. NanoESI-MS experiments have demonstrated that, in contrast to QTRT2, wild type Z. mobilis TGT exists almost entirely as homodimer with no significant amount of monomer being detectable.16 Crystal structure analyses of Z. mobilis TGT variants with reduced dimer stability due to mutations in the aromatic cluster show the accommodation of water molecules in the dimer interface.60 The observation of a water molecule next to the aromatic cluster in the interface of the QTRT2 dimer supports our hypothesis that penetrating water molecules indicate increasing destabilisation of dimer contact. In bacterial TGT, a strictly conserved glutamate (Glu339´ in Z. mobilis TGT) within the short loop connecting helices αE and αF is able to form a salt bridge to a lysine (Lys52 in Z. mobilis TGT), which is located in loop β1´α1´ (i.e. in the loop-helix motif) of the dimer mate and invariant as well (Figures S10A and S11A). In QTRT2, the position equivalent to Glu339´ of Z. mobilis TGT is also occupied by a glutamate (Glu372´). However, Lys52 of the bacterial enzyme is replaced by a proline (Pro43) in QTRT2, which is not able to form a salt bridge to Glu372´ (Figures S10D and S11C). This may be a further reason for the reduced stability of the QTRT2 homodimer in comparison to the bacterial TGT homodimer. Mutation of Lys52 in the Z. mobilis enzyme to a sterically similar but uncharged methionine was shown to significantly affect the conformation of the loop-helix motif leading to decreased dimer stability and enzymatic activity16,59 (Figure S10B).

Homology model of QTRT1 and its heterodimer protein-protein interface with QTRT2 It is unknown why bacteria utilise a homodimeric TGT while a heterodimeric enzyme, consisting of a catalytic and a noncatalytic subunit, has evolved in eukaryotes. However, in a previous study we have shown that mutational adaptation of the substrate base binding pocket of a bacterial TGT ACS Paragon Plus Environment

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towards that of QTRT1 is not sufficient to create an enzyme which is able to insert queuine into tRNA.18 The crystal structure of a correspondingly mutated Z. mobilis TGT variant determined in complex with queuine shows that the electron density assigned to this base does by no means correlate with the nominal resolution of the structure. Instead, it appears fuzzy and lacks detail with the dihydroxy-cyclopentenyl moiety not being defined at all. This indicates that queuine is only loosely bound to the active centre and does not adopt a well-defined binding pose. In contrast, in the crystal structure of this TGT variant in complex with preQ1, the ligand, which is identically positioned as in the wild-type enzyme, is excellently defined. This suggests that queuine may constitute a more demanding substrate for TGT than preQ1, although the underlying reasons are hardly understood. Accordingly, the turnover of queuine may require the best possible orientation of the tRNA, especially of nucleotide 34, towards the active site. Possibly, the appearance of a noncatalytic subunit, specialised in binding and positioning the bulk body of the tRNA substrate, may have helped to overcome this challenge. In contrast, bacterial TGT, which does not use queuine as a substrate base, can manage with a homodimeric enzyme that requires only one tgt gene within the streamlined prokaryotic genome. Clearly, the high resolution crystal structure of a QTRT1/QTRT2 heterodimer in complex with a tRNA substrate will be necessary to understand the advantage of a heterodimeric over a homodimeric TGT in eukaryotes. Furthermore, there are no clues as to why QTRT2 forms a relatively stable homodimer while QTRT1 does not. It is tempting to speculate that QTRT2 homodimer formation may have a regulatory role but, so far, there is no real evidence for such a function. To obtain understanding about the instability of the QTRT1 homodimer as well as of the high stability of the QTRT1/QTRT2 heterodimer we created in-silico models of both complexes. To this end, we used the crystal structure of T. maritima TGT as a template for the generation of the murine QTRT1 model (for details see the Materials and Methods section).


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The model of the QTRT1 homodimer reveals that the aromatic cluster within the dimer interface is significantly smaller than that observed in bacterial TGTs or in the QTRT2 homodimer. As in T. maritima TGT and in QTRT2, the bicyclic indole of Trp326 of Z. mobilis TGT is replaced by a monocyclic aromatic ring system (His325 in QTRT1). In contrast to T. maritima TGT and QTRT2, however, there is no nearby tyrosine compensating for the smaller size of this aromatic side chain. Tyr302 of T. maritima TGT and Tyr354 of QTRT2 (Figure 4) correspond to Pro320 in QTRT1, which makes no vander-Waals contact to His325 (Figure S12A). While Phe92´ of Z. mobilis TGT (Phe79´ in T. maritima TGT and Phe84´ in QTRT2) is conserved in QTRT1 (Phe95´), Tyr330 of Z. mobilis TGT (Tyr311 in T. maritima TGT and Tyr363 in QTRT2) is replaced by Phe329. Although analysis with DrugScore-PPI suggests that this residue makes the largest contribution to dimer stability, it lacks the hydroxyl group of tyrosine and, accordingly, is not able to form an H-bond with a residue of loop β1´α1´ as observed in the crystal structures of Z. mobilis TGT, T. maritima TGT and murine QTRT2. Strikingly, the aromatic side chain of His333 of Z. mobilis TGT (His314 in T. maritima TGT and His366 in QTRT2) is absent in QTRT1. His333 is replaced by an alanine residue (Ala332), whose side chain methyl group merely forms a van-der-Waals contact to the methyl group of Ala52´ (Figure S12A). An additional residue that, according to the analysis of our model with DrugScore-PPI, provides an important contribution to QTRT1 homodimer formation is Asn338 within loop αEαF. Via its side chain carboxamide it forms three H-bonds to the main chain carbonyl oxygens of Gln51’, Met54’ and Gly56’ (Figure S11D). This extensive interaction with residues of loop β1´α1´, however, does not compensate for the truncated aromatic cluster in this subunit, as reflected by the low tendency of QTRT1 to form a homodimer. Interestingly, in QTRT1, Asn338 takes the position of Glu339 in Z. mobilis TGT, which is invariant in bacterial TGTs and able to form a subunit bridging ionic interaction with Lys52´ (Z. mobilis TGT numbering) (Figures S11A and S11B). Since the QTRT1/QTRT2 heterodimer lacks the two-fold rotational symmetry inherent to homodimeric TGTs, its dimer interface consists of two structurally different halves and, therefore, con-


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tains two different versions of the aromatic cluster. The core of the first version is composed of aromatic residues His359, Tyr363 and His366 from QTRT2 as well as of Phe95 from QTRT1 (Figure S12B). As observed in the interface of the QTRT2 homodimer it is expanded by Tyr354 from QTRT2, which makes, however, no H-bond to any residue of QTRT1 in our model. In contrast, Tyr363 (QTRT2) forms an H-bond to the main chain carbonyl of Ala52 (QTRT1), which is equivalent to the H-bond formed by Tyr330 and Ala49´ in Z. mobilis TGT and by Tyr311 and Ala36´ in T. maritima TGT (Figures 4A and 4B). In a previous study, we had shown that this interaction significantly contributes to the stability of the bacterial TGT dimer.16 According to the results of DrugScore-PPI analysis, the H-bond between Tyr363 (QTRT2) and Ala52 (QTRT1) is energetically slightly more favourable than that formed by Tyr311 and Ser41´ in the homodimer interface of QTRT2 (Figure 4C). His366 (QTRT2), whose counterparts in bacterial TGTs of known structure make an H-bond to the main chain carbonyl of a residue within loop β1´α1´, is not involved in such a polar interaction. Instead, it is embedded in a hydrophobic environment composed of the side chains of QTRT1 residues Leu77, Pro81, Leu85 and Phe95. Analysis with DrugScore-PPI suggests a major contribution of these Van-der-Waals contacts to heterodimer stability. In the immediate vicinity of the described aromatic hot spot, our model displays an inter-subunit salt bridge formed by Glu372 (QTRT2) and Lys55 (QTRT1) (Figure S11E), which corresponds to that formed by Glu339 and Lys52´ in Z. mobilis TGT or Glu320 and Lys39´ in T. maritima TGT (Figures S11A and S11B). The core of the second aromatic cluster within the heterodimer interface of our model consists of QTRT1 residues Phe329 and His325 and of the QTRT2 residue Phe84 (Figure S12C). The fourth aromatic (histidine) residue, normally involved in the formation of the aromatic hot spot core, is missing. Instead, the corresponding position is taken by an alanine (Ala332 of QTRT1). The gain of space caused by the absence of the imidazole, however, allows the phenyl moiety of Phe329 (QTRT1) to move into the resulting void. This, in turn, allows the phenol group of Tyr77 (QTRT2) to change its position in such a way, that it can make both a van-der-Waals contact to Phe84 (QTRT2) and an Hbond to the side chain hydroxyl of Ser326 (QTRT1) (Figure S12C). Due the large hydrophobic contact ACS Paragon Plus Environment

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area gained by this conformational change and the newly formed H-bond, Tyr77 (QTRT2) provides an important contribution to heterodimer stability as calculated by DrugScore-PPI. Furthermore, as observed in the homodimer interface of the QTRT2 crystal structure, His47 (QTRT2) and His70 (QTRT2) expand the aromatic cluster and, thus, further compensate for the missing histidine. A salt bridge corresponding to that formed by Glu372 (QTRT2) and Lys55 (QTRT1) near the first aromatic cluster in the QTRT1/QTRT2 dimer interface is not observed close to the second aromatic hot spot. Here, the position of Glu372 (QTRT2) is taken by Asn338 (QTRT1) while Lys55 (QTRT1) is replaced by Pro43 (QTRT2). Clearly, the side chains of these residues are not able to form any ionic interaction. Instead, Asn338 (QTRT1) forms, via its side chain carboxamide, an H-bond to the side chain hydroxyl group of Ser41 within loop β1α1 of QTRT2 (Figure S11F). In future studies, site directed mutagenesis experiments are required to assess and quantify the significance of the described individual interactions in matters of respective dimer stabilities. In addition, the crystal structures of QTRT1 and of the QTRT1/QTRT2 heterodimer are prerequisite to confirm or disprove the details observed in our in-silico models. Until then, the latter provide valuable and plausible hints about the diverging stabilities of different dimeric TGT (subunit) complexes. The understanding of these, in turn, is central to developing compounds which specifically disturb the bacterial TGT dimer while leaving the human enzyme unaffected.


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Funding This work was supported by the “Agence Nationale de la Recherche” (ANR) and the “French Proteomic Infrastructure” (ProFI; ANR-10-INBS-08-03). GIS IBiSA and Région Alsace allocated financial support in purchasing a Synapt G2 HDMS instrument. The PhD fellowships of S. Petiot-Bécard and T. Botzanowski were funded by NovAliX and the “Institut de Recherche Servier”, respectively. A travel grant was provided by the “Helmholz-Zentrum für Materialien und Energie” in Berlin.

Supporting Information Figures S1 – S12 (PDF), Homology models of the QTRT1 homodimer and the QTRT1/QTRT2 heterodimer (PDB)

Acknowledgements We would particularly like to acknowledge the help and support of Christian Feiler and Martin Gerlach during the experiment at BESSY II for providing outstanding support. We thank Christian Sohn for his help during “in house” X-ray data collection, Stefanie Dörr for expert technical assistance and Ralf Pöschke from MARXTAL facility for performing the initial crystallisation screen.


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Biochemistry

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For Table of Contents Use Only Homodimer architecture of QTRT2, the noncatalytic subunit of the eukaryotic tRNA-guanine transglycosylase Christina Behrens, Inna Biela, Stéphanie Petiot-Bécard, Thomas Botzanowski, Sarah Cianférani, Christoph P. Sager, Gerhard Klebe, Andreas Heine, Klaus Reuter


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Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the

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