Structure of Trna-Modifying Enzyme Tias and Motions of Its Substrate Binding Zinc Ribbon

Article

Structure of tRNA-Modifying Enzyme TiaS and Motions of Its Substrate Binding Zinc Ribbon

Jianshu Dong 1,2,3,4,6,10, Fahui Li 6, Feng Gao 6, Jia Wei 6, Yajing Lin 6, Yong Zhang 6, Jizhong Lou 6, Guangfeng Liu 9, Yuhui Dong 8, Lin Liu 7, Hongmin Liu 3, Jiangyun Wang 6 and Weimin Gong 5,6,

1 - School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China 2 - Institute of Drug Discovery and Development, Zhengzhou University, Zhengzhou 450001, PR China 3 - Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education of China, Zhengzhou University, Zhengzhou 450001, PR China 4 - Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou University, Zhengzhou 450001, PR China 5 - Hefei National Laboratory for Physical Science at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 6 - Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 7 - Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 8 - Beijing Synchrotron Radiation Facility and Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China 9 - Shanghai Synchrotron Radiation Facility and Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China 10 - University of Chinese Academy of Sciences, Beijing 100864, China

Correspondence to Jianshu Dong and Weimin Gong: J. Dong is to be contacted at: School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR China.; W. Gong, is to be contacted at: Hefei National Laboratory for Physical Science at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China. [email protected]; [email protected] https://doi.org/10.1016/j.jmb.2018.08.015 Edited by Yigong Shi

Abstract

The accurate modification of the tRNAIle anticodon wobble cytosine 34 is critical for AUA decoding in protein synthesis. Archaeal tRNAIle2 cytosine 34 is modified with agmatine in the presence of ATP by TiaS (tRNAIle2 agmatidine synthetase). However, no structure of apo-form full-length TiaS is available currently. Here, the crystal structures of apo TiaS and a complex of TiaS–agmatine–AMPPCP–Mg are presented, with properly folded zinc ribbon and Cys4-zinc coordination identified. Compared with tRNAIle2-bound form, the architecture of apo TiaS shows a totally different conformation of zinc ribbon. Molecular dynamics simulations of the docking complex between free-state TiaS and tRNAIle2 suggest that zinc ribbon domain is capable of performing large-scale motions to sample substrate binding-competent conformation. Principle component analysis and normal mode analysis show consistent results about the relative directionality of functionally correlated zinc ribbon motions. Apo TiaS and TiaS–agmatine–AMPPCP–Mg/TiaS–AMPCPP–Mg complex structures capture two snapshots of the flexible ATP-Mg binding p2loop step-by-step stabilization. Research from this study provides new insight into TiaS functional mechanism and the dynamic feature of zinc ribbons. © 2018 Elsevier Ltd. All rights reserved.

Introduction cytosine 34 (Cyt34) is crucial for AUA deciphering in both bacteria and archaea [3–7]. In eukaryotes, the Of the more than 1 hundred currently known naturally modified 34th-base pseudouridine or inosine is occurring post-transcriptional modifications, tRNA responsible for recognition of the third-base adenine contains the greatest chemical diversity [1,2].The of the AUA codon [8,9]. For most bacteria [10],Tils modification of tRNAIle2 anticodon wobble position (tRNAIle2 lysidine synthetase) modifies tRNAIle2 with

0022-2836/© 2018 Elsevier Ltd. All rights reserved. J Mol Biol (2018) 430, 4183–4194 4184 tRNA-Modifying Enzyme TiaS lysine, thus changing both the tRNA identity and significant effects on the agmatine modification activity, specificity for codon recognition [4,11–18]. Without but mutation with the acceptor stem of tRNAIle2 lysine modification, precursor of tRNAIle is charged substituted by that of tRNAMet abolishes the activity. with methionine rather than isoleucine. Recently, AUA While the deletion of ZRD seems to have little impact on decoding system is uncovered in some Archaea phyla, ATP hydrolysis activity, tRNAIle2 phosphorylation and namely, Euryoarchaeota, Crenarchaeota and Thau- agmatination activities are sharply reduced. Also, point marchaeota [19–21]. Agmatine, a polyamine also mutations of residues at TiaS ZRD (R369A, or C352A– found in the mammalian nervous system [22,23],is C355A double mutation) severely impair agmatine utilized to modify tRNAIle2 Cyt34, by joining the amino incorporation activity. However, another ZRD point group of agmatine with the 2′ carbon of the pyrimidine mutation, E360A, unexpectedly increases agmatidine ring; the resulted new nucleotide agmatidine or agm2C yield. is found in Haloarcula marismortui, Methanococcus Previously, crystal structures of TiaS at three different maripaludis, Sulfolobus solfataricus, Sulfolobus states have been successfully solved (TiaS–tRNAIle2– tokodaii, Archaeoglobus fulgidus and some other ATP, TiaS–tRNAIle2–AMPCPP–agmatine and TiaS archaea [19–21]. After modification, the third tertiary lacking ZRD with agmatine), which suggest the amine group of the pyrimidine ring is protonated to form molecular basis of agm2C formation [25]. However, secondary amine group and can then donate instead of the fourth domain of TiaS essential for substrate tRNA accept proton in hydrogen bonding. The amine group at selection is still largely uncharacterized, and no C4 position is converted to imino group. The modified structural information of apo full-length TiaS enzyme tRNAIle2 could then decode AUA rather than AUG is available so far. Previously, we have reported full- codon via agmatidine34–adenosine unorthodox base length TiaS enzyme structure without substrate tRNA pairing [19–21,24]. As a consequence, the mature but with AMPPCP and click-reactive compound N-(4- tRNAIle is isoleucylated, while the unmodified tRNAIle aminobutyl)-2-azidoacetamide (AGN) bound at the precursor is methionylated [19], which highlights the agmatine binding pocket, revealing the structural significance of the modification. The enzyme catalyzing basis of site- and sequence-specific covalent labeling this modification is named tRNAIle2 agmatidine synthe- of RNAs in mammalian cells (4RVZ.pdb) [29],butZRD tase (TiaS). and the functional significance have not been dis- Catalytic mechanism of tRNAIle2 agmatidine syn- cussed. Here, the crystal structures of apo full-length thesis is uncovered recently through structural and TiaS with intact fourth domain at 2.5-Å resolution and a biochemical studies of TiaS–tRNAIle2 complex complex of TiaS–agmatine–AMPPCP–Mg without [25–28]. TiaS is found to be able to modify tRNAIle2 tRNA are presented, with Cys4-zinc coordination and with the presence of agmatine and ATP without fourth domain zinc ribbon identified. Zinc ribbon/finger adenylated intermediate. TiaS is a four-domain motifs are prevalent in various protein families, which protein; the N-terminal domain responsible for ATP play key roles in a variety of life processes [30–38], binding is named Thr18–Cyt34 kinase domain mediating protein–protein, protein–DNA or protein– (TCKD), as TiaS is able to phosphorylate both its RNA interactions. Various kinds of residue combina- own conservative Thr18 and the tRNAIle2 Cyt34 for tions (C2H2, C4 or C2HC, etc.) are employed to chelate activation. The second and third domains are named zinc atom [38–40], with C2H2 zinc finger regarded as ferredoxin-like fold (FLD) and OB fold (OBD), respec- the classical. ZRD recognizes the acceptor arm of tively. Extensive site-directed mutagenesis study tRNAIle2 in a base-specific manner [25].HereTiaS also supports the functional assignment of the enzyme crystal structures present different conforma- first three domains as the enzyme catalytic core tions from tRNA-bound state. The functionally correlat- [21,25,26]. The anticodon loop of tRNAIle2 binds to ed direction preferences of zinc ribbon motions upon the inter-domain cavity of the TiaS enzyme core. TiaS– substrate binding uncovered from molecular dynamics tRNAIle2–ATP and TiaS–tRNAIle2–AMPCPP– and normal mode analysis (NMA) reveal an amazing agmatine complex structures show similar conforma- new attribute of zinc ribbons, providing fresh insight into tion of TiaS enzyme. tRNAIle2 recognition and modification mechanism. The The C terminus of TiaS, which is responsible for implications for applied research on targetable protein tRNAIle2 acceptor arm recognition and substrate engineering are discussed as well. selection, is named zinc ribbon domain (ZRD) domain – for its similarity to the zinc ribbon-like fold [25,26].TiaS Results tRNAIle2 structures show that ZRD interacts with the major groove of the tRNAIle2 acceptor arm. Because of the presence of two other kinds of tRNA (the initiator Archaeal TiaS structure and its zinc ribbon and elongator tRNAsMet) in the cell that bear the same anticodon loop sequence, TiaS should discriminate The crystal structure of free full-length archaeal tRNAIle2 precursor from other tRNAs like tRNAMet by AfTiaS was determined at 2.5-Å resolution (Fig. 1, recognizing acceptor stem regions [25].Replacingthe Table 1). X-ray fluorescence analysis suggested the anticodonstemoftRNAIle2 with that of tRNAMet has no presence of zinc element in TiaS enzyme crystals. tRNA-Modifying Enzyme TiaS 4185

Fig. 1. Structure of archaeal TiaS. (a) Overall structure of TiaS with properly folded ZRD (ZRD). (b) 2Fo − Fc electron density map (blue mesh) of ZRD contoured at 1σ. (c) Electrostatic surface of ZRD. (d) Coordination of zinc by four cysteine ligands, 2Fo − Fc map of Cys4-zinc contoured at 1.2σ (blue) and at 6σ (cyan). (e) Superposition of TiaS ZRD and zinc ribbon of DNA binding protein PRIA (4 nl4.pdb, TiaS in magenta and PRIA in silver color). (f) Superposition of TiaS ZRD and zinc ribbon of RNA polymerase II (5iy6.pdb, TiaS in magenta and RNA polymerase in blue color).

Primary sequence alignment of TiaS enzyme from zinc ribbons from RNA polymerase II and primo- different Archaea showed the presence of four some assembly protein PRIA (a DNA binding invariant cysteine residues (Fig. S1). In addition, protein) [41–43] (Fig. 1e, f). Three-dimensional the fourth domain of TiaS enzyme exhibited a typical structural alignment of ZRD with these similar Cys4 zinc ribbon structure (Figs. 1a, b and S2), and zinc zinc knuckles showed that RMSDs of Cαs were ribbon was connected by an extended antiparallel β as little as 1 Å (Fig. S2). Like other classical zinc sheet to the core enzyme consisting of the first three ribbons, AfTiaS ZRD also contained a three- domains, as also suggested by previous studies stranded antiparallel β-sheet in the structure, and [25]. Electrostatic surface of ZRD showed large the two β-hairpins both contributed for zinc binding. areas of electropositive region, which was in AfTiaS ZRD showed left-handed geometry, and the agreement with its function as being responsible majority of the zinc ribbon structures adopt this for tRNAIle2 acceptor stem recognition and binding geometry [38]. The TiaS zinc ribbon structure (Fig. 1c). represented here was actually in sharp contrast to Properly folded Cys4 zinc ribbon was interpreted the substrate tRNAIle2-bound form. from the electron density maps (Fig. 1b, d). The highly conserved four cysteine residues of TiaS Apo TiaS showed a different conformation of responsible for zinc atom chelation were Cys352, zinc ribbon Cys355, Cys370 and Cys373, which were contributed by two zinc knuckles (Fig. 1d). The zinc–ligand The crystal structure of apo TiaS showed a bond lengths were 2.4, 2.5, 2.4 and 2.6 Å, which different conformation from the tRNAIle2-bound were consistent with typical cysteine–sulfur zinc state. The overall structures of the core N-terminal bond length obtained from structurally known zinc three domains of TiaS were similar; however, large- finger motifs (roughly within the range of 2.0~2.6 Å) scale conformational changes of the zinc ribbon [39]. And ICP-MS analysis indicated that zinc was were observed (Fig. 2a, b). Actually, ZRD confor- the major transition metal present in the pure protein mations in all the crystal structures of TiaS enzyme sample. DaliLite structural search of AfTiaS ZRD from this study were highly similar (Fig. S3), indicated that the closest structural relatives were including the TiaS–agmatine–AMPPCP–Mg and 4186 tRNA-Modifying Enzyme TiaS

Table 1. Statistics of data collection and model refinement tRNA-bound state. Free zinc ribbon presented an of TiaS selenomethione derivative and native structures extended properly folded structure with outer edge α α Native Se-Met distance from Gly356C to Arg374C about 12.8 Å away (Fig. 2e). To validate the crystallographic Data collection Nov-2010 April-2011 observations, SAXS experiment was performed by Space group P41212 P41212 Cell dimensions using free wild-type native TiaS protein sample, a, b, c (Å) 69, 69, 211 70, 70, 211 and small-angle X-ray scattering profile of TiaS α, β, γ (°) 90 90 protein in solution strongly favors our crystallographic a Resolution (Å) 50–2.5 (2.56–2.47) 30–3.0 (3.11–3.00) model over the TiaS model from TiaS–tRNA complex Reflections 18,850 (1852) 11,149 (1073) b (3AMT.pdb, Fig. S4, Table S4). Rsym or Rmerge 0.090 (0.474) 0.086 (0.487) I/σI 21.3 (3.8) 53 (11) Completeness (%) 99.1 (99.1) 99.9 (100.0) Motions of substrate binding zinc ribbon by Redundancy 11.7 (9.5) 42.2 (42.3) molecular dynamics Refinement Resolution (Å) 40–2.5 No. reflections 18,539 The structural differences suggested that TiaS c Rwork/Rfree 0.244/0.292 experienced drastic conformational changes upon No. atoms substrate tRNAIle2 binding. To test this hypothesis, Protein 3282 isolated substrate tRNAIle2 (3AMT.pdb) was docked to Ligand/ion 2 B-factors full-length TiaS structure from this study, and the Protein 88 complex was subjected to molecular dynamics simu- Ligand/ion 91 lation in explicit water. Consistent with results dis- R.m.s deviations cussed above, the least dynamical regions were the Bond lengths (Å) 0.008 enzyme core (Fig. 3a, b), while the largest root mean Bond angles (°) 1.221 Ramachandran plot square fluctuations were located in or near the ZRD. It Most favored regions 93.7% basically showed a moderate rigid body motion of the Additionally allowed 4.9% ZRD. The conformational dynamics arose from Generously allowed 1.5% twisting and stretching motions of the loop region a Numbers in parentheses represent the value for the highest- linkers relative to the core enzyme (Fig. 3c, e); further resolution shell. stretching and twisting of these linkers resulted in ZRD b ∑ − ∑ Ile2 Rmerge = |Ii Im |/ Ii, where Ii is the intensity of the measured relocation binding onto tRNA acceptor arm, as also reflection and Im is the mean intensity of all symmetry-related reflections. observed from the changing distance between ZRD c Ile2 Rcryst = σ||Fobs | − |Fcalc ||/σ|Fobs |, where Fobs and Fcalc are and tRNA and between Tyr185 and Gly356 along observed and calculated structure factors. Rfree = σT ||Fobs | − the simulation (Fig. 3d). The non-conserved linkers σ |Fcalc ||/ T |Fobs |, where T is a test data set of about 5% of the total located at basically the same position in different reflections randomly chosen and set aside prior to refinement. Archael TiaS enzymes and variable length of ZRD linkers could be observed from the primary sequences (Fig. S1). To further understand TiaS ZRD dynamics and to selenomethionine-substituted structures. Compared examine whether the motions were random or had with tRNAIle2-bound form, TiaS X-ray structures preferred directionality, principal component analysis here showed that ZRD was much further away from (PCA) was performed using the simulation trajectory substrate (Fig. 2a, b), Gly356Cα movedlongdistance (Figs. 3e and S5). The first and second principal (about 14 Å) away from tRNAIle2 binding direction. The components accounted for 44% and 13% of the overall new conformation enabled ZRD to establish interac- variance, respectively, and the first three components tion with the antiparallel β sheet connecting ZRD and together accounted for 62%. In other words, the first two OBD (Fig. 2c). A least-squares superposition of the Cα principal components contributed more than 50% to atoms of both states was performed to reveal the functionally important transition between the free backbone displacement (Fig. 2d). Large-scale motions and the binding competent states. NMA was also of Arg364Cα and Cys373Cα, about 11 and 12 Å performed using TiaS structure from this study, with sideward, respectively, were also observed. There more than 90% contribution of the first three modes to was an up to 18-degree rotation for the whole ZRD in the experimentally observed direction of ZRD motions total (Fig. 2b). The distances between Gly356Cα of the (Figs. 3f and S6), and molecular dynamic simulation, ZRD and Tyr185Cα were about 48 Å for apo TiaS or PCA and NMA showed consistent results about the TiaS–agmatine–AMPPCP–Mg structures and 59 Å for relative amplitude and directionality of the motions of tRNAIle2-bound TiaS, and the two zinc knuckles TiaS ZRD. together with the chelated zinc atom of TiaS were Although ZRD was involved in crystal packing in the well ordered for all the structures from this study TiaS, TiaS–agmatine–AMPPCP–Mg, TiaS–AMPCPP– (Figs. 1b,dandS3).Therelativepositionandthe Mg and previously reported TiaS–tRNAIle2–ATP struc- shape of TiaS zinc ribbon were both different from tures (3AMT.pdb), the direction of ZRD movement tRNA-Modifying Enzyme TiaS 4187

Fig. 2. Conformational difference of TiaS zinc ribbon between free (magenta) and tRNAIle2-bound form (cyan). (a) Compared to substrate tRNAIle2-bound form, free TiaS showed that ZRD moved away from tRNAIle2 acceptor stem. (b) Superposition of TiaS and tRNAIle2-bound TiaS showed large-scale motions of ZRD (about 18° rotation); Gly356Cα, Arg364Cα and Cys373Cα moved about 14, 11 and 12 Å, respectively. (c) For free TiaS ZRD, Ile351 was within van der Waals' interaction distance from Leu381 (3.9 Å), and Arg358 formed hydrogen bonds with Asn349. (d) Backbone displacement of TiaS relative to the tRNAIle2-bound form. (e) TiaS zinc knuckle from this study was structured. made it unlikely that ZRD conformational change and tRNAIle2 anticodon loop bound to TiaS from two observed here was resulted from crystal packing sides of p2loop, and p2loop (including Y51-R54) main (Fig. S7). To bind substrate tRNAIle2,ZRDmoved chain and side chains both had been modeled for and bent toward the acceptor stem to establish the TiaS–tRNAIle2–ATP structure [25], which meant interaction (Figs. 2aand3c, d), as revealed both that p2loop was structurally poised in this situation experimentally and computationally. Crystal structures (3AMT.pdb). Therefore, these structures just captured of TiaS–tRNAIle2 complex contained buried interface different stabilization states of ATP-Mg binding p2loop area of 1.98 × 103 Å2 between the enzyme and (Fig. 4). In fact, p2loop was conserved in most archaeal substrate tRNA, of which about 0.50 × 103 Å2 in- TiaS proteins (Fig. S1). Compared to simulations of volved the ZRD. During the simulation, tRNAIle2–TiaS docked TiaS–tRNAIle2, root mean square fluctuations hadanextendedinterfaceareaofupto2.10× 103 Å2, of apo TiaS during simulation suggested the high of which about 0.65 × 103 Å2 involved ZRD, while the flexibility of free p2loop (Fig. 4e) as well, which was in interface area in the docked complex was approxi- agreement with crystallographic temperature factor mately 1.59 × 103 Å2, with ZRD contributing about analysis (Fig. S8). 0.11 × 103 Å2. There were large areas of electronegative region at the ATP triphosphate binding site formed by D8, D9, Motions of ATP–Mg binding p2loop D11, Tpo18 and G57 (Fig. S9), and TiaS AMPPCP interaction was mediated through Mg2+ (Fig. 4f). TiaS Another conformational difference from substrate bind to and hydrolyze ATP with the presence of Mg2+. Ile2 tRNA -bound TiaS came from ATP binding p2loop. Thedissociationconstant(Kd) between ATP and No electron density was observed for residues Y51– TiaS determined by ITC in the presence of Mg2+ was T53 of p2loop in the apo TiaS structure, which 63 ± 9.7 μM, but this affinity decreased about indicated their flexibility (Fig. 4a), and all three complex 1000-fold without Mg2+ (Fig. 4g). TiaS protein was structures of TiaS–agmatine–AMPPCP–Mg, TiaS– able to hydrolyze ATP here in the presence of Mg2+, AMPCPP–Mg (at 2.7 Å) and previously reported but this activity was abolished without Mg2+ under the TiaS–AMPPCP–AGN structure (4RVZ.pdb) were same conditions (Fig. S10). The significance of Mg2+- highly similar to the structure of apo TiaS except mediated interaction between β,γ-phosphates and p2loop (Fig. S3, Table S1). All showed p2loop (Y51– TiaS was also supported by the observation that the T53) main chain (Fig. 4b–d). Comparison of 4RVZ and affinity between AMP-Mg/AMP and TiaS was trivial thecurrentstructurewasshowninFig.S3.ATP-Mg compared to that of ATP-Mg and TiaS (Fig. S11). 4188 tRNA-Modifying Enzyme TiaS

Fig. 3. Motions of TiaS by molecular dynamics simulation and NMA. The simulation was started from a docking complex of free TiaS and tRNAIle2. (a) Root mean square fluctuations (RMSF) of Cα atoms during simulations. (b) Backbone RMSD during the simulation relative to the starting crystal structure of the free state. (c) One snapshot (yellow) from the trajectory was superimposed onto structures of free state TiaS (magenta) and substrate tRNAIle2-bound complex (3AMT, TiaS in cyan, and tRNAIle2 in gray). The conformational changes were a result of twisting and stretching motions around four linkers, indicated by arrows (same as panel e). (d) Distance fluctuation between TiaS ZRD and tRNAIle2 (top) and that of Tyr185–Gly356 during the simulation (bottom). (e, f) Relative amplitudes and directions of ZRD motions were visualized by the direction and size of the gray ellipsoids, (e) between the most dissimilar structures calculated by PCA using Cα position covariance from the simulation, and (f) motions of TiaS calculated by NMA.

AccordingtobothTiaS–agmatine–AMPPCP–Mg and direction (Figs. 2aand3). Computationally, ZRD TiaS–AMPCPP–Mg structures, the side chains of D8, samples substrate binding-competent conformation D9, D11, Tpo18, I143, and I49, N56 from p2loop were within nanoseconds. ZRD conformational change is responsible for ATP-Mg recognition (Fig. 4); this was functionally correlated, because without these archi- further examined through site-directed mutagenesis tectural adaptations, ZRD will not bind onto the experiment. As ITC showed, the affinities between tRNAIle2 acceptor arm, which is required for tRNAIle2 ATP-Mg and these mutants decreased significantly agmatination catalyzed by TiaS. Encoded in the compared with that of wild type (Fig. 4, Table 2). primary sequence of the enzyme, the large-scale Thereby, Mg2+ ions were required for TiaS to bind and motions of ZRD may be induced by substrate tRNAIle2 hydrolyze ATP, like many WalkerB ATP binding binding, as crystallography, molecular dynamic sim- proteins, although TiaS TCKD was dissimilar to other ulation, PCA and NMA results shown about the known ATP binding proteins. direction preference of TiaS ZRD motions (Fig. 3). To summarize, this study shows functionally correlated large-scale motions of zinc ribbon of a full-length Discussion active enzyme. TiaS enzyme core and ZRD are both essential for Here, crystal structure of free TiaS reveals properly substrate tRNAIle2 binding, and TiaS associates with folded ZRD, which is in sharp contrast to tRNAIle2- tRNAIle2 by recognizing not only acceptor stem bound TiaS with respect to conformations. Preliminary regions but also anticodon stem and loop. Previous SAXS experiment indicates that free TiaS in solution study suggested that ZRD instead of the enzyme favors conformation observed from this crystallo- core firstly interacts with substrate tRNAIle2, regard- graphic study. To form TiaS–tRNAIle2 complex, ZRD ing the binding route [25]. But in fact, the enzyme preferentially moves toward the substrate binding core provides the major binding energy to hold tRNA-Modifying Enzyme TiaS 4189

Fig. 4. ATP binding p2loop of TiaS. 2Fo − Fc map (blue) contoured at 1σ, Fo − Fc omit map (green) contoured at 3σ. (a) Apo TiaS, no electron density observed for residues Y51–T53 of p2loop. (b) TiaS–agmatine–AMPPCP–Mg complex. (c) TiaS–AMPCPP–Mg complex. Y51–R54 main chains were traced for both panels b and c. (d) Superposition of TiaS p2loop. Apo TiaS in magenta, TiaS–agmatine–AMPPCP–Mg in yellow, TiaS–AMPCPP–Mg in green, and TiaS-ATP- tRNAIle2 in cyan. (e) Comparison of p2loop RMSF during simulation between free TiaS and TiaS–tRNAIle2 complex. (f) AMPPCP–Mg binding profile to TiaS, residues were shown as sticks. (g) ATP-Mg, ATP relative affinities to TiaS. (h) Affinities between ATP-Mg and TiaS mutants normalized to that of wt-TiaS. substrate tRNAIle2 in place (Table. S2). As discussed as AfTiaS (Fig. S12). Results from this study lead us to above, the interface area between enzyme core and propose that an alternative route of binding between tRNAIle2 is about two times larger than that of entire TiaS and tRNAIle2 may be possible; that is, TiaS ZRD, and the enzyme core accounts for about two enzyme core firstly binds substrate tRNAIle2,and thirds of the interaction energy between TiaS and tRNAIle2 anticodon loop is loaded into the catalytic tRNAIle2 (Table. S2). There is another tRNA modifier cavity for initial recognition, then ZRD performs showing highly similar enzyme catalytic core structure conformational change to bind acceptor stem for subsequent recognition. For many zinc ribbon containing proteins, nucleic acid binding is usually Table 2. The dissociate constant (Kd value) obtained from achieved by the strand present between the two zinc ITC in this study knuckles. This is the case for TiaS, and this strand interacts with the antiparallel linker in the free state, Protein Ligand Kd (μM) while the rotation and bending motion of the antipar- WtTiaS ATP-Mg 63 ± 9.7 allel linker breaks up this interaction and establishes D8A 694 ± 26 Ile2 D9A 328 ± 23 new interactions between this strand and the tRNA D11A 667 ± 21 acceptor arm, although the tRNA is not a passive T18A 376 ± 18 participant during the recognition and binding pro- N56A 595 ± 23 cess. MD simulation reveals moderate motion of the I49R 575 ± 38 – I143R 256 ± 15 ZRD. The strand (i.e., Met359 Arg364) of TiaS ZRD is I143K 571 ± 26 firstly tethered to the edge of the acceptor stem, and D8A–D9A 1571 ± 86 then the strand mounts onto the tRNA major groove D9A–D11A 962 ± 37 (Fig. 3). TiaS ZRD and tRNA acceptor stem are pulled D8A–D9A–D11A 2370 ± 130 WtTiaS ATP 7891 ± 180 closer to each other primarily by electrostatic attrac- WtTiaS Agmatine 85 ± 5.7 tion, as revealed by electrostatic surface analysis of ZRD (Fig. 1c) and molecular dynamic simulations. ITC results between ATP-Mg and site-directed mutants of TiaS were ZRD is positively charged and tRNA sugar-phosphate obtained in 25 mM Hepes (pH 7.9), 0.5 M NaCl and 1 mM DTT backbone are negatively charged at normal physio- buffer, with wild-type TiaS as positive control (Kd =67±5.2μM). ITC between ATP-Mg and wild-type TiaS in 25 mM Hepes (pH 7.9), logical conditions. To summarize, an alternative 50 mM NaCl and 1 mM DTT buffer gave similar value of Kd (63 ± mechanism of function regarding the initial recognition μ 9.7 M). Kd between agmatine and wild-type TiaS was obtained in and binding process is proposed in Fig. 5, adding to 25 mM Hepes (pH 7.9), 50 mM NaCl and 1 mM DTT buffer. the previously proposed catalytic mechanism [25]. 4190 tRNA-Modifying Enzyme TiaS

Fig. 5. Proposed two-step model for recognition and binding of substrate tRNA by TiaS enzyme. The first step shows initial interaction of the two macromolecules free in solution through long-range electrostatic attractions. Recognition and binding of tRNA anticodon region by shape-complementary TiaS enzyme core are driven by the decrease of enthalpy and release of free energy, and a strong association between the two is achieved after this step (Table S2). In the second step, dynamical TiaS ZRD performs conformational change and binds onto the acceptor stem of substrate tRNA. Cooperative binding of tRNA and ATP-Mg2+ to TiaS is suggested here. The last step within a catalytic cycle is proposed here showing how TiaS prepares itself for another modifying reaction.

Compared to the model proposed earlier, this study they rarely undergo scaffold unfolding releasing zinc suggests a different answer to the question of which upon binding their target partner. part binds first. TiaS enzyme core firstly binds onto Research from this study provides new insight into tRNA anticodon region for initial recognition. The the conformational dynamics of zinc ribbons and zinc interaction between ZRD and the acceptor stem is the ribbon linkers, which may aid in zinc finger engineering last and critical discrimination step for substrate [49–51]. Substrate tRNAIle2 acceptor stem is recog- selection, and this interaction will probably outlive nized by ZRD in a nucleoside-specific manner, and the interaction between anticodon region and the four consecutive nucleotides 5′-CCCA-3′ are recog- enzyme core, because unlike the anticodon loop, nized by four residues Glu360, Ser361, Ala362 and nothing changed here after agmatination. The un- Gln366 (Fig. S13). The mode of nucleic acid recogni- modified precursor of tRNAIle2 will eventually compete tion by TiaS ZRD is thus principally a one-to-one away mature tRNAIle2 when the anticodon region of interaction between individual amino acids from the the former occupies the enzyme core. The binding of recognition zinc ribbon to individual RNA bases substrate tRNA and ATP-Mg2+ to TiaS is probably in a (Fig. S13), and this precise interaction pattern is an cooperative manner, as binding by either one would important feature required for zinc finger engineering stabilize the p2loop facilitating the binding of the other. [30]. Also, the two guanosine bases, which base pair Concerning how TiaS releases AMP and gets ready with the middle two cytosines of 5′-CCCA-3′,are for another cycle of modification, the affinities between recognized by Arg369. Analysis of the molecular TiaS and AMP or AMP-Mg2+ are measured through dynamic modes indicated that ZRD is able to sample a ITC, and it is found that they are very weak (Fig. S11). broad distribution of conformations, indicating that Therefore, the release of AMP is probably an zinc ribbon is designed to provide its substrates with a automatic process. large set of conformations, probably for more swift Zinc finger conformational changes upon partner recognition. The dynamic and flexible feature of binding have been reported before [44,45], and the zinc ribbon linkers discovered here may help the binding of substrate RNA induces the folding of the design of targetable proteins with enhanced versatility second zinc finger of tristetraprolin in solution [46]. sterically—increasing affinity and specificity to target Also, drastic conformational change of ID1 (insertion nucleic acid while adding more zinc ribbon/finger domain 1, a zinc ribbon) of the dimeric glycyl building blocks. Targetable nucleases (using zinc tRNA synthetase was observed, although the zinc fingers or CRISPR/Cas) [31,52] engineering had wide ribbon like structure of ID1 did not preserve the 4 applications ranging from research to medicine cysteine residues or zinc atom in either states [47,48]. [30,50]. New characteristics about zinc ribbon discov- Zinc-ribbon/finger motifs are relatively stable struc- ered from this study may help future designs of tures constrained by limited bond length variability to targetable proteins for applications in gene expression coordinate the zinc atom with cognate residues, and regulation, genome manipulation or cancer therapy. tRNA-Modifying Enzyme TiaS 4191

Materials and Methods increase of DNA ratio and the addition of 2 mM DTT (dithiothreitol) into the protein sample combined with the increase of NH4Ac salt concentration in the TiaS protein purification and crystallization crystallization condition produced crystals about 0.1 mm and diffracted to 4 Å. The 7-nt DNA fragment TiaS gene AF2259 from A. fulgidus was cloned into with the sequence of 5′-CTCATAA-3′ was mixed with pET22b and pET28a vectors between Nde1 and Xho1 TiaS at a molar ratio of 3:1 for crystallization. Crystals restriction sites, for the production of C-terminal His×6 grown by sitting drop vapor diffusion in 50 mM Hepes- tagged (for TiaS-pET22b) and dual His tagged (for Na (pH 7.0~7.2), 0.5 M NH4Ac,0.2MMgCl2 and TiaS-pET28a) protein, respectively. The C-terminal 1.5%–2.5% PEG8000 for about 10 days at 16 °C were His tagged TiaS was arbitrarily chosen during later used for diffraction data collection. Selenomethionine- stages of the project. TiaS-pET22b recombinant substituted TiaS was expressed in methionine auxo- plasmid vector was transformed into BL21 (DE3)- trophe cell line B834 (DE3), purified in the same competent cells. Target proteins were overexpressed procedure, crystallized at 50 mM Mes (pH 6.7), 0.1 M at 36 °C after induction by 1 mM IPTG for 3.5 h. The NH4Ac, 0.02 M MgCl2 and 2% PEG8000. TiaS– harvested cell pellet was resuspended in 50 mM agmatine–AMPPCP–Mg and TiaS–AMPCPP–Mg Hepes-Na (pH 7.9), 0.5 M NaCl and 10% glycerol complex crystals were obtained from co-crystallization. and stored at −30 °C. Cells were disrupted by All the TiaS protein samples used here were as sonication on ice in 50 mM Hepes-Na (pH 7.9), isolated from host Escherichia coli cells, which was 0.5 M NaCl, 10% glycerol, 5 mM beta-mercaptoetha- quite similar with reported methods. For mutagenesis, nol and 1 mM PMSF. After centrifugation at 4 °C for please see supplementary information for detail. 50 min, target protein in the supernatant was captured by Nickel column at room temperature. The Nickel Data collection and structure determination resin was firstly washed by lysis buffer and then was thoroughly washed by 50 mM Hepes-Na (pH 7.9) and Diffraction data were collected at the wavelength 1 M NaCl. Target protein was obtained from gradient of 0.979 Å at Shanghai Synchrotron Radiation imidazole elution. Protein sample was concentrated Facility (SSRF) BL17U with Q315CCD detector. and applied to gel filtration using superdex200 column The phase angle was resolved by single-wavelength with 20 mM Tris–HCl (pH 8) and 0.05 M NaCl as anomalous diffraction (Fig. S14) strategy using running buffer at 4 °C. Then the protein sample was selenomethionine derivative. Data processing and concentrated and flash-frozen in liquid nitrogen and reduction were carried out using the HKL2000 stored at −80 °C for further experiments. Initial package [53]. Heavy atom positions were obtained crystallization experiments with either C terminal from SHELX C/D. TiaS native structure was solved His-tagged or dual His-tagged protein at either 4 °C by molecular replacement using Se-TiaS as search or 16 °C failed to produce any crystal. The full-length model. Structure models were refined with maximum TiaS from Methanococcus jannaschii showed negligi- likelihood protocols using REFMAC, CNS and ble expression after induction. Truncated TiaS (1–407 Phenix [54–56]. TiaS–agmatine–AMPPCP–Mg and and 108–420) showed decreased solubility. Heat TiaS–AMPCPP–Mg structures were solved by mo- treatment at 50 °C or 60 °C on the full-length AfTiaS lecular replacement using native apo-TiaS as search protein did not improve purity or homogeneity. Then model. No DNA was observed in any of the solved ATP analogue AMPPCP, agmatine, and tRNAIle2 electron density map. Crystallography statistics anticodon loop analogue DNA fragment in various were summarized in Tables 1 and S1. Superposition lengths were added to protein sample both separately of structures was performed using UCSF chimera and in alternative combinations before screening [57]. Cartoon and other structure representations crystallization kits. The first crystal was observed were generated using UCSF chimera or PyMOL from dual His×6-tagged TiaS enzyme supplemented [57,58]. with 7-nt DNA fragment (CTCATAA) synthesized from Sangon. TiaS was diluted to 10 mg/ml and mixed with Isothermal titration calorimetry measurements the DNA at a molar ratio of 1:1.2; crystals appear a week later in 0.1 M NH4Ac, 0.02 M MgCl2,0.05M All ITC experiments were carried out using ITC200 Hepes sodium (pH 7.0) and 5% w/v PEG8000. (Micro Cal) at 20 °C. Wild-type proteins were titrated Both C-terminal His-tagged and dual His-tagged by agmatine, ATP-Mg and AMP-Mg in the 25 mM TiaS enzyme could crystallize at this condition. Hepes-Na (pH 7.9), 50 mM NaCl and 1 mM DTT The C-terminal His-tagged TiaS was arbitrarily chosen buffer. Each titration experiment consisted of one for the following optimization. Variation of pH and 0.4-μl (duration time, 0.8 s) injection followed by precipitant PEG8000 concentration failed to grow nineteen 2-μl (duration time, 4 s) injections. Ligands crystals bigger, and change of precipitants to (1.5 mM) in the syringe were used to titrate 0.1 mM PEG200, PEG400, PEG600, PEG3350, PEG4000 or TiaS in the cell, with 1.5 mM ligands in the syringe PEG6000 did not improve the situation, while the titrating buffer as control. 4192 tRNA-Modifying Enzyme TiaS

TiaS ATP hydrolysis activity assay Chen, Peng Wu, Peng Xue, etc.) for assistance in crystallography, SAXS, mutagenesis, ITC or MS Enzchek pyrophosphate assay kit from Invitrogen measurement. We thank Yuxiao Wang and Daniel was used to examine ATP hydrolysis activity of TiaS, Hoffmann. This project was supported by the Ministry either wild type or mutants. The reaction mixture of Science and Technology of China (grant number containing 0.5 μMTiaSand0.5mMATPwas 2011CB910503), National Science Foundation of incubated at 50 °C for 1 h before applied to pyrophos- China (grant number 91219202) and Zhengzhou phate/phosphate detection using spectrophotometer University young teacher special fund to J.D. (grant as suggested by the protocol. number 32210872).

Molecular dynamics simulation and NMA Appendix A. Supplementary data Molecular dynamics simulation was carried out by NAMD [59] using CHARMM all-atom force field Supplementary data to this article can be found [60–62]. Full-length TiaS structure from this study was online at https://doi.org/10.1016/j.jmb.2018.08.015. docked to tRNAIle2 (from 3AMT.pdb, [25]) as rigid body using DOT2.0 [63]. The interface area between TiaS Received 16 January 2018; Ile2 3 2 and tRNA was 1.6 × 10 Å in the docked complex, Received in revised form 2 August 2018; and the shortest distance between any non-hydrogen Accepted 7 August 2018 Ile2 atoms of TiaS ZRD and tRNA was more than 3.7 Å Available online 16 August 2018 away. The complex was used for simulation in explicit TIP3P water inside an orthorhombic box with a 15-Å Keywords: padding in each direction. The system was neutralized zinc finger; with 50 mM NaCl. The final system contained 96,758 conformational change; atoms. Particle Mesh Ewald was used to treat long- tRNA modification; range electrostatic interactions, and they were shifted catalytic mechanism to be zero beyond 12 Å. Bond lengths involving hydrogen atoms were fixed by the SHAKE algorithm. Abbreviations used: Thetimestepwas2fs.Theproductionrunwas Cyt34, cytosine 34; TiaS, tRNAIle2 agmatidine synthetase; performed at 300 K for 10 ns. The simulation was ZRD, zinc ribbon domain; PCA, principal component repeated with a different starting model—the TiaS– analysis; NMA, normal mode analysis. tRNA complex crystal structure to valid the observations (3AMT, with selenium atoms being replaced by sulfur atoms and the antiparallel linker and ZRD being References replaced by that of the apo TiaS from this study; Fig. S18). PCA (also called quasi-harmonic) of the molecular dynamics trajectory was performed by [1] W.A. Cantara, P.F. Crain, J. Rozenski, J.A. McCloskey, K.A. diagonalizing the atom fluctuation cross-correlation Harris, X. Zhang, et al., The RNA modification database, matrix using Bio3D [64]. The overall translational and RNAMDB: 2011 update, Nucleic Acids Res. 39 (2011) rotational motions in the trajectory were eliminated by D195–D201. least squares fitting to the first frame. [2] M.A. Machnicka, K. Milanowska, O. Osman Oglou, E. Purta, NMA of full-length TiaS was performed using both M. Kurkowska, A. Olchowik, et al., MODOMICS: a database of RNA modification pathways—2013 update, Nucleic Acids Anisotropic Network Model [65] and Elastic Network – Model [66], and they showed similar results. Res. 41 (2013) D262 D267. [3] F.H. Crick, Codon–anticodon pairing: the wobble hypothesis, J. Mol. Biol. 19 (1966) 548–555. Data access [4] T. Muramatsu, K. Nishikawa, F. Nemoto, Y. Kuchino, S. Nishimura, T. Miyazawa, et al., Codon and amino-acid Coordinates and structure factors have been depos- specificities of a transfer RNA are both converted by a single ited in the Protein Data Bank (5XOB and 6AGG). post-transcriptional modification, Nature 336 (1988) 179–181. [5] G.R. Björk, Biosynthesis and function of modified nucleosides, tRNA: Structure, Biosynthesis, and Function, American Society for Microbiology, Washington, DC 1995, pp. 165–205. [6] S. Yokoyama, NS, Modified nucleosides and codon recognition, tRNA: Structure, Biosynthesis, and Function, Acknowledgments American Society for Microbiology, Washington, DC 1995, pp. 207–224. We thank Prof Yanli Wang, Dr. Mingzhu Wang, [7] T. Suzuki, Biosynthesis and function of tRNA wobble modifi- Weiqiong Gan and the staff members in BL17U- cations, in: H. Grosjean (Ed.), Fine-Tuning of RNA Functions SSRF, BSRF and the protein science platform at IBP by Modification and Editing, Springer, Berlin Heidelberg 2005, (Fuquan Yang, Zhensheng Xie, Xiang Ding, Yuanyuan pp. 23–69. tRNA-Modifying Enzyme TiaS 4193

[8] B. Senger, S. Auxilien, U. Englisch, F. Cramer, F. Fasiolo, [25] T. Osawa, S. Kimura, N. Terasaka, H. Inanaga, T. Suzuki, T. The modified wobble base inosine in yeast tRNAIle is a Numata, Structural basis of tRNA agmatinylation essential positive determinant for aminoacylation by isoleucyl-tRNA for AUA codon decoding, Nat. Struct. Mol. Biol. 18 (2011) synthetase, Biochemistry 36 (1997) 8269–8275. 1275–1280. [9] Marcus J.O. Johansson, A.S. Bystro, Transfer RNA [26] N. Terasaka, S. Kimura, T. Osawa, T. Numata, T. Suzuki, modifications and modifying enzymes in Saccharomyces Biogenesis of 2-agmatinylcytidine catalyzed by the dual cerevisiae, in: H. Grosjean (Ed.) Fine-Tuning of RNA protein and RNA kinase TiaS, Nat. Struct. Mol. Biol. 18 Functions by Modification and Editing, 12, Springer, New (2011) 1268–1274. York 2005, pp. 87–119. [27] T. Osawa, H. Inanaga, S. Kimura, N. Terasaka, T. Suzuki, T. [10] T. Taniguchi, K. Miyauchi, D. Nakane, M. Miyata, A. Muto, S. Numata, Crystallization and preliminary X-ray diffraction Nishimura, et al., Decoding system for the AUA codon by analysis of an archaeal tRNA-modification enzyme, TiaS, tRNAIle with the UAU anticodon in mycoplasma mobile, complexed with tRNA(Ile2) and ATP, Acta Crystallogr. F Nucleic Acids Res. 41 (2013) 2621–2631. Struct. Biol. Cryst. Commun. 67 (2011) 1414–1416. [11] F. Harada, S. Nishimura, Purification and characterization of [28] T. Numata, Mechanisms of the tRNA wobble cytidine AUA specific isoleucine transfer ribonucleic acid from modification essential for AUA codon decoding in prokary- Escherichia coli B, Biochemistry 13 (1974) 300–307. otes, Biosci. Biotechnol. Biochem. 79 (2015) 347–353. [12] T. Muramatsu, S. Yokoyama, N. Horie, A. Matsuda, T. Ueda, [29] F. Li, J. Dong, X. Hu, W. Gong, J. Li, J. Shen, et al., A Z. Yamaizumi, et al., A novel lysine-substituted nucleoside in covalent approach for site-specific RNA labeling in mamma- the first position of the anticodon of minor isoleucine tRNA lian cells, Angew. Chem. Int. Ed. Engl. 54 (2015) 4597–4602. from Escherichia coli, J. Biol. Chem. 263 (1988) 9261–9267. [30] A. Klug, The discovery of zinc fingers and their applications in [13] A. Soma, Y. Ikeuchi, S. Kanemasa, K. Kobayashi, N. gene regulation and genome manipulation, Annu. Rev. Ogasawara, T. Ote, et al., An RNA-modifying enzyme that Biochem. 79 (2010) 213–231. governs both the codon and amino acid specificities of [31] N.P. Pavletich, C.O. Pabo, Zinc finger-DNA recognition: isoleucine tRNA, Mol. Cell 12 (2003) 689–698. crystal structure of a Zif268–DNA complex at 2.1 Å, Science [14] Y. Ikeuchi, A. Soma, T. Ote, J. Kato, Y. Sekine, T. Suzuki, 252 (1991) 809–817. Molecular mechanism of lysidine synthesis that determines [32] L. Fairall, J.W. Schwabe, L. Chapman, J.T. Finch, D. Rhodes, tRNA identity and codon recognition, Mol. Cell 19 (2005) The crystal structure of a two zinc-finger peptide reveals an 235–246. extension to the rules for zinc-finger/DNA recognition, Nature [15] K. Nakanishi, S. Fukai, Y. Ikeuchi, A. Soma, Y. Sekine, T. 366 (1993) 483–487. Suzuki, et al., Structural basis for lysidine formation by ATP [33] Y. Shi, J.M. Berg, Specific DNA–RNA hybrid binding by zinc pyrophosphatase accompanied by a lysine-specific loop and finger proteins, Science 268 (1995) 282–284. a tRNA-recognition domain, Proc. Natl. Acad. Sci. U. S. A. [34] D. Su, Z. Lou, F. Sun, Y. Zhai, H. Yang, R. Zhang, et al., 102 (2005) 7487–7492. Dodecamer structure of severe acute respiratory syndrome [16] H. Grosjean, G.R. Bjork, Enzymatic conversion of cytidine coronavirus nonstructural protein nsp10, J. Virol. 80 (2006) to lysidine in anticodon of bacterial isoleucyl-tRNA—an 7902–7908. alternative way of RNA editing, Trends Biochem. Sci. 29 [35] J. Lu, M. Sun, K. Ye, Structural and functional analysis of (2004) 165–168. Utp23, a yeast ribosome synthesis factor with degenerate [17] K. Nakanishi, L. Bonnefond, S. Kimura, T. Suzuki, R. Ishitani, PIN domain, RNA 19 (2013) 1815–1824. O. Nureki, Structural basis for translational fidelity ensured by [36] S.A. Wolfe, L. Nekludova, C.O. Pabo, DNA recognition by transfer RNA lysidine synthetase, Nature 461 (2009) Cys2His2 zinc finger proteins, Annu. Rev. Biophys. Biomol. 1144–1148. Struct. 29 (2000) 183–212. [18] M. Kuratani, Y. Yoshikawa, Y. Bessho, K. Higashijima, T. [37] R.S. Brown, Zinc finger proteins: getting a grip on RNA, Curr. Ishii, R. Shibata, et al., Structural basis of the initial binding of Opin. Struct. Biol. 15 (2005) 94–98. tRNA(Ile) lysidine synthetase TilS with ATP and L-lysine, [38] S.S. Krishna, I. Majumdar, N.V. Grishin, Structural classification Structure 15 (2007) 1642–1653. of zinc fingers: survey and summary, Nucleic Acids Res. 31 [19] C. Kohrer, G. Srinivasan, D. Mandal, B. Mallick, Z. Ghosh, J. (2003) 532–550. Chakrabarti, et al., Identification and characterization of a [39] S. Karlin, Z.Y. Zhu, Classification of mononuclear zinc metal tRNA decoding the rare AUA codon in Haloarcula marismortui, sites in protein structures, Proc. Natl. Acad. Sci. U. S. A. 94 RNA 14 (2008) 117–126. (1997) 14231–14236. [20] D. Mandal, C. Kohrer, D. Su, S.P. Russell, K. Krivos, C.M. [40] J.H. Laity, B.M. Lee, P.E. Wright, Zinc finger proteins: new Castleberry, et al., Agmatidine, a modified cytidine in the insights into structural and functional diversity, Curr. Opin. anticodon of archaeal tRNA(Ile), base pairs with adenosine Struct. Biol. 11 (2001) 39–46. but not with guanosine, Proc. Natl. Acad. Sci. U. S. A. 107 [41] L. Holm, L.M. Laakso, Dali server update, Nucleic Acids Res. (2010) 2872–2877. 44 (2016) W351–W355. [21] Y. Ikeuchi, S. Kimura, T. Numata, D. Nakamura, T. Yokogawa, [42] L. Holm, S. Kaariainen, P. Rosenstrom, A. Schenkel, T. Ogata, et al., Agmatine-conjugated cytidine in a tRNA Searching protein structure databases with DaliLite v.3, anticodon is essential for AUA decoding in archaea, Nat. Bioinformatics 24 (2008) 2780–2781. Chem. Biol. 6 (2010) 277–282. [43] K.D. Westover, D.A. Bushnell, R.D. Kornberg, Structural [22] D.J. Reis, S. Regunathan, Is agmatine a novel neurotransmitter basis of transcription: nucleotide selection by rotation in the in brain? Trends Pharmacol. Sci. 21 (2000) 187–193. RNA polymerase II active center, Cell 119 (2004) 481–489. [23] A. Halaris, J. Plietz, Agmatine: metabolic pathway and [44] A.I. Arunkumar, G.C. Campanello, D.P. Giedroc, Solution spectrum of activity in brain, CNS Drugs 21 (2007) 885–900. structure of a paradigm ArsR family zinc sensor in the [24] T. Suzuki, T. Numata, Convergent evolution of AUA decoding DNA-bound state, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) in bacteria and archaea, RNA Biol. 11 (2014) 1586–1596. 18177–18182. 4194 tRNA-Modifying Enzyme TiaS

[45] E. Bochkareva, S. Korolev, S.P. Lees-Miller, A. Bochkarev, system: a new software suite for macromolecular structure Structure of the RPA trimerization core and its role in the determination, Acta Crystallogr. D Biol. Crystallogr. 54 (1998) multistep DNA-binding mechanism of RPA, EMBO J. 21 905–921. (2002) 1855–1863. [57] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. [46] P.J. Blackshear, W.S. Lai, E.A. Kennington, G. Brewer, G.M. Greenblatt, E.C. Meng, et al., UCSF Chimera—a visualization Wilson, X. Guan, et al., Characteristics of the interaction of a system for exploratory research and analysis, J. Comput. synthetic human tristetraprolin tandem zinc finger peptide Chem. 25 (2004) 1605–1612. with AU-rich element-containing RNA substrates, J. Biol. [58] The PyMOL Molecular Graphics System, LLC, Schrödinger, Chem. 278 (2003) 19947–19955. 2008, Version 1.2. [47] X. Qin, Z. Hao, Q. Tian, Z. Zhang, C. Zhou, W. Xie, Cocrystal [59] J.C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, structures of glycyl-tRNA synthetase in complex with tRNA E. Villa, et al., Scalable molecular dynamics with NAMD, suggest multiple conformational states in glycylation, J. Biol. J. Comput. Chem. 26 (2005) 1781–1802. Chem. 289 (2014) 20359–20369. [60] A.D. MacKerell, D. Bashford, M. Bellott, R.L. Dunbrack, J.D. [48] G. Kaur, S. Subramanian, The insertion domain 1 of class IIA Evanseck, M.J. Field, et al., All-atom empirical potential for dimeric glycyl-tRNA synthetase is a rubredoxin-like zinc molecular modeling and dynamics studies of proteins, ribbon, J. Struct. Biol. 190 (2015) 38–46. J. Phys. Chem. B 102 (1998) 3586–3616. [49] F.D. Urnov, E.J. Rebar, M.C. Holmes, H.S. Zhang, P.D. [61] R. Wu, Z. Lu, Z. Cao, Y. Zhang, A transferable nonbonded Gregory, Genome editing with engineered zinc finger pairwise force field to model zinc interactions in metallopro- nucleases, Nat. Rev. Genet. 11 (2010) 636–646. teins, J. Chem. Theory Comput. 7 (2011) 433–443. [50] D. Carroll, Genome engineering with targetable nucleases, [62] T. Zhu, X. Xiao, C. Ji, J.Z.H. Zhang, A new quantum Annu. Rev. Biochem. 83 (2014) 409–439. calibrated force field for zinc–protein complex, J. Chem. [51] H. Kim, J.S. Kim, A guide to genome engineering with Theory Comput. 9 (2013) 1788–1798. programmable nucleases, Nat. Rev. Genet. 15 (2014) 321–334. [63] V.A. Roberts, E.E. Thompson, M.E. Pique, M.S. Perez, L.F. [52] H. Zhao, G. Sheng, J. Wang, M. Wang, G. Bunkoczi, W. Gong, Ten Eyck, DOT2: macromolecular docking with improved et al., Crystal structure of the RNA-guided immune surveillance biophysical models, J. Comput. Chem. 34 (2013) 1743–1758. cascade complex in Escherichia coli, Nature 515 (2014) [64] B.J. Grant, A.P. Rodrigues, K.M. Elsawy, J.A. McCammon, 147–150. L.S. Caves, Bio3d: an R package for the comparative [53] Z. Otwinowski, W. Minor, Processing of X-ray diffraction data analysis of protein structures, Bioinformatics 22 (2006) collected in oscillation mode, Methods Enzymol. 276 (1997) 2695–2696. 307–326. [65] E. Eyal, G. Lum, I. Bahar, The anisotropic network model web [54] The CCP4 suite: programs for protein crystallography, Acta server at 2015 (ANM 2.0), Bioinformatics 31 (2015) Crystallogr. D Biol. Crystallogr. 50 (1994) 760–763. 1487–1489. [55] P.D. Adams, P.V. Afonine, G. Bunkoczi, V.B. Chen, I.W. [66] E. Lindahl, C. Azuara, P. Koehl, M. Delarue, NOMAD-Ref: Davis, N. Echols, et al., PHENIX: a comprehensive Python- visualization, deformation and refinement of macromolecular based system for macromolecular structure solution, Acta structures based on all-atom normal mode analysis, Nucleic Crystallogr. D Biol. Crystallogr. 66 (2010) 213–221. Acids Res. 34 (2006) W52–W56. [56] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. Delano, P. Gros, R.W. Grosse-Kunstleve, et al., Crystallography & NMR