Structure-based insights into the mechanism of [4Fe-4S]-dependent sulfur insertase LarE.

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP-dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe-4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster-free (apo-) and cluster-bound (holo-) forms. The structure of holo-MmLarE shows that the [4Fe-4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein-bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo-MmLarE and the previously reported biochemical investigations of holo-LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe-5S] intermediate, and transfers it to the substrate, as in the case of [4Fe-4S]-dependent tRNA thiolation enzymes.

The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis.

Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 Å resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

Iron-sulfur biology invades tRNA modification: the case of U34 sulfuration.

Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron-sulfur [Fe-S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe-4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe-S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.

Structural Evidence for a [4Fe-5S] Intermediate in the Non-Redox Desulfuration of Thiouracil.

We recently discovered a [Fe-S]-containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Šresolution is reported; it harbors a [4Fe-4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4-thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non-protein-bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe-5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site-directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non-redox enzymatic desulfuration of thiouracil, in which a [4Fe-4S] cluster binds and activates the sulfur atom of the substrate.

Structure-based mechanistic insights into catalysis by tRNA thiolation enzymes.

In all domains of life, ribonucleic acid (RNA) maturation includes post-transcriptional chemical modifications of nucleosides. Many sulfur-containing nucleosides have been identified in transfer RNAs (tRNAs), such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), 2-methylthioadenosine (ms2A). These modifications are essential for accurate and efficient translation of the genetic code from messenger RNA (mRNA) for protein synthesis. This review summarizes the recent discoveries concerning the mechanistic and structural characterization of tRNA thiolation enzymes that catalyze the non-redox substitution of oxygen for sulfur in nucleosides. Two mechanisms have been described. One involves persulfide formation on catalytic cysteines, while the other uses a [4Fe-4S] cluster, chelated by three conserved cysteines only, as a sulfur carrier.

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster.

Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a protein-bound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.

An extended dsRBD is required for post-transcriptional modification in human tRNAs.

In tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upregulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis. Here, we report the crystal structure of the individual domains of HsDus2 and their functional characterization. HsDus2 is organized into three major modules. The N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved α-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). Enzymatic assays and yeast complementation showed that the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. While in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the α-helical domain, we showed that in HsDus2 this function is carried out by the dsRBD. This is the first reported case of a tRNA-modifying enzyme carrying a dsRBD used to bind tRNAs.

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase.

In most organisms, the widely conserved 1-methyl-adenosine58 (m1A58) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m1A57 being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A57 but not A58, confirming that PabTrmI methylates efficiently the first adenine of the A57A58A59 sequence. PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m1A58 formation triggers RNA release. A model of the protein-tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.

The crystal structure of wild-type human brain neuroglobin reveals flexibility of the disulfide bond that regulates oxygen affinity.

Neuroglobin plays an important function in the supply of oxygen in nervous tissues. In human neuroglobin, a cysteine at position 46 in the loop connecting the C and D helices of the globin fold is presumed to form an intramolecular disulfide bond with Cys55. Rupture of this disulfide bridge stabilizes bi-histidyl haem hexacoordination, causing an overall decrease in the affinity for oxygen. Here, the first X-ray structure of wild-type human neuroglobin is reported at 1.74 Šresolution. This structure provides a direct observation of two distinct conformations of the CD region containing the intramolecular disulfide link and highlights internal cavities that could be involved in ligand migration and/or are necessary to enable the conformational transition between the low and high oxygen-affinity states following S-S bond formation.

FAD/folate-dependent tRNA methyltransferase: flavin as a new methyl-transfer agent.

RNAs contain structurally and functionally important modified nucleosides. Methylation, the most frequent RNA modification in all living organisms, mostly relies on SAM (S-adenosylmethionine)-dependent methyltransferases. TrmFO was recently discovered as a unique tRNA methyltransferase using instead methylenetetrahydrofolate and reduced flavin adenine dinucleotide (FAD) as essential cofactors, but its mechanism has remained elusive. Here, we report that TrmFO carries an active tRNA-methylating agent and characterize it as an original enzyme-methylene-FAD covalent adduct by mass spectrometry and a combination of spectroscopic and biochemical methods. Our data support a novel tRNA methylating mechanism.

The human tRNA m (5) C methyltransferase Misu is multisite-specific.

The human tRNA m ( 5) C methyltransferase Misu is a novel downstream target of the proto-oncogene Myc that participates in controlling cell division and proliferation. Misu catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosines in tRNAs. It was previously shown to catalyze in vitro the intron-dependent formation of m ( 5) C at the first position of the anticodon (position 34) within the human pre-tRNA (Leu) (CAA). In addition, it was recently reported that C48 and C49 are methylated in vivo by Misu. We report here the expression of hMisu in Escherichia coli and its purification to homogeneity. We show that this enzyme methylates position 48 in tRNA (Leu) (CAA) with or without intron and positions 48, 49 and 50 in tRNA (Gly2) (GCC) in vitro. Therefore, hMisu is the enzyme responsible for the methylation of at least four cytosines in human tRNAs. By comparison, the orthologous yeast enzyme Trm4 catalyzes the methylation of carbon 5 of cytosine at positions 34, 40, 48 or 49 depending on the tRNAs.

Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

Structural comparison of tRNA m(1)A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions.

BACKGROUND: tRNA m(1)A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m(1)A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers. RESULTS: In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges. CONCLUSIONS: The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.

Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis.

The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton.

Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase.

The S-adenosyl-L-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m(1)A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m(1)A also at the adjacent position 57, m(1)A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m(1)A57/58 methyltransferase ((Pab)TrmI), in complex with S-adenosyl-L-methionine or S-adenosyl-L-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of (Pab)TrmI. When A59 is absent in tRNA(Asp), only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that (Pab)TrmI methylates the first adenine of an AA sequence.

Pseudo-merohedral twinning in monoclinic crystals of wild-type human brain neuroglobin.

The purification, crystallization and successful structure determination by molecular replacement of wild-type human brain neuroglobin at 1.8 A resolution is reported. The apparent space group was orthorhombic C222(1), but the real space group was monoclinic P2(1), which resulted from twinning. Indeed, the unit-cell parameters, a = 31.2, b = 139.1, c = 31.2 A, beta = 102 degrees , display a fortuitously close to c and twinning by the operator l, -k, h occurs. Twinning was not evident from the initial analysis of intensity distribution, but pseudo-merohedral twinning was revealed by the Padilla and Yeates test based on local intensity differences. A twinning fraction of 0.5 was determined in SHELXL, indicating a perfect hemihedrally twinned crystal. To date, this type of twinning has been reported in more than ten structures, which makes it quite a common case in proteins.

The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity.

The 5-methyluridine is invariably found at position 54 in the TPsiC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase ((Pab)TrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of (Pab)TrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 A resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of (Pab)TrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by (Pab)TrmU54. These results were used to model yeast tRNA(Asp) in the (Pab)TrmU54 structure to get further insights into the different RNA specificities of RumA and (Pab)TrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to (Pab)TrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TPsiC loop conformational change has to occur for the flipping of the target uridine into the (Pab)TrmU54 active site during catalysis.

Crystal structure of Thermus thermophilus tRNA m1A58 methyltransferase and biophysical characterization of its interaction with tRNA.

Methyltransferases from the m(1)A(58) tRNA methyltransferase (TrmI) family catalyze the S-adenosyl-l-methionine-dependent N(1)-methylation of tRNA adenosine 58. The crystal structure of Thermus thermophilus TrmI, in complex with S-adenosyl-l-homocysteine, was determined at 1.7 A resolution. This structure is closely related to that of Mycobacterium tuberculosis TrmI, and their comparison enabled us to enlighten two grooves in the TrmI structure that are large enough and electrostatically compatible to accommodate one tRNA per face of TrmI tetramer. We have then conducted a biophysical study based on electrospray ionization mass spectrometry, site-directed mutagenesis, and molecular docking. First, we confirmed the tetrameric oligomerization state of TrmI, and we showed that this protein remains tetrameric upon tRNA binding, with formation of complexes involving one to two molecules of tRNA per TrmI tetramer. Second, three key residues for the methylation reaction were identified: the universally conserved D170 and two conserved aromatic residues Y78 and Y194. We then used molecular docking to position a N(9)-methyladenine in the active site of TrmI. The N(9)-methyladenine snugly fits into the catalytic cleft, where the side chain of D170 acts as a bidentate ligand binding the amino moiety of S-adenosyl-l-methionine and the exocyclic amino group of the adenosine. Y194 interacts with the N(9)-methyladenine ring, whereas Y78 can stabilize the sugar ring. From our results, we propose that the conserved residues that form the catalytic cavity (D170, Y78, and Y194) are essential for fashioning an optimized shape of the catalytic pocket.