The structure of the TrmE GTP-binding protein and its implications for tRNA modification
2004; Springer Nature; Volume: 24; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7600507
ISSN1460-2075
AutoresAndrea Scrima, Ingrid R. Vetter, M.‐Eugenia Armengod, Alfred Wittinghofer,
Tópico(s)Metal-Organic Frameworks: Synthesis and Applications
ResumoArticle16 December 2004free access The structure of the TrmE GTP-binding protein and its implications for tRNA modification Andrea Scrima Andrea Scrima Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany Search for more papers by this author Ingrid R Vetter Ingrid R Vetter Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany Search for more papers by this author M Eugenia Armengod M Eugenia Armengod Insituto de Investigationes Citológicas, Fondación Valenciana de Investigationes Biomédicas, Valencia, Spain Search for more papers by this author Alfred Wittinghofer Corresponding Author Alfred Wittinghofer Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany Search for more papers by this author Andrea Scrima Andrea Scrima Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany Search for more papers by this author Ingrid R Vetter Ingrid R Vetter Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany Search for more papers by this author M Eugenia Armengod M Eugenia Armengod Insituto de Investigationes Citológicas, Fondación Valenciana de Investigationes Biomédicas, Valencia, Spain Search for more papers by this author Alfred Wittinghofer Corresponding Author Alfred Wittinghofer Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany Search for more papers by this author Author Information Andrea Scrima1, Ingrid R Vetter1, M Eugenia Armengod2 and Alfred Wittinghofer 1 1Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany 2Insituto de Investigationes Citológicas, Fondación Valenciana de Investigationes Biomédicas, Valencia, Spain *Corresponding author. Max-Planck Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. Tel.: +49 231 133 2100; Fax: +49 231 133 2199; E-mail: [email protected] The EMBO Journal (2005)24:23-33https://doi.org/10.1038/sj.emboj.7600507 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal α/β domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate. Introduction TrmE is a member of the guanine nucleotide-binding proteins (GNBP), which bind and hydrolyse GTP. It contains a canonical G domain and is conserved in all three kingdoms of life. Normally, G-domain proteins cycle between a GTP-bound state, which represents the active state of the protein, and an inactive, GDP-bound state. The activation and inactivation of GNBP is further controlled by guanine nucleotide exchange factors (GEFs). GEFs catalyse the exchange of GDP to GTP and thereby activate the protein. GTPase activating proteins (GAPs) accelerate the slow intrinsic hydrolysis rate. In contrast to the family of Ras-like small and the heterotrimeric large G proteins, which regulate many crucial cellular processes like differentiation, cell–cell adhesion and nuclear and vesicular transport by 'switching' signalling pathways on and off, TrmE is believed to be directly involved in an enzymatic reaction, the modification of the wobble position uridine (U34) in tRNAs in bacteria, yeast and mammalia (i.e. tRNALys, tRNAGlu, tRNA4Leu, tRNA4Arg and probably tRNAGln). Bacterial strains lacking the 50 kDa TrmE protein are deficient in the biosynthesis of tRNA modified at position 5 (Elseviers et al, 1984). TrmE-assisted modification of U34 at the 5 position of the uridine base leads to 5-methylaminomethyl-uridine (mnm5U) in bacteria, 5-carboxymethylaminomethyl-uridine in yeast and 5-taurinomethyl-uridine in human. The modification at U34 allows interaction with G and A, but restricts base pairing with C and U (Yokoyama et al, 1979; 1985; Yokoyama and Nishimura, 1995). This is extremely important in mixed codon box families (Glu, Gln, Lys, Leu and Arg) for which base pairing of U with C or U would lead to misincorporation of amino acids. Furthermore, the modification influences frameshifting during the translation process (Brierley et al, 1997; Hagervall et al, 1998; Bjork et al, 1999; Urbonavicius et al, 2001). The modification of U34 requires many different proteins: MnmA catalyses the thiolation of U34 at the 2 position leading to s2U (Sullivan et al, 1985). In the modification pathway, TrmE (also called MnmE), together with the protein GidA (Elseviers et al, 1984; Brégeon et al, 2001), is believed to be involved in the addition of the cmnm group at the 5 position, although the precise role of both proteins in the modification reaction is unknown. In a following modification step, the TrmC protein is believed to catalyse the formation of mnm5U (Hagervall et al, 1987) (Figure 1). The modifications at the 5 and the 2 position of uridine are independent of each other, and thiolation of U34 has been performed in vitro with recombinant proteins (MnmA and IscS) (Lauhon, 2002; Kambampati and Lauhon, 2003). Figure 1.Proposed pathway for the biosynthesis of mnm5U at U34 in tRNA. TrmE and GidA are postulated to be involved in the first modification step for the biosynthesis of 5-carboxymethylaminomethyl-uridine (Elseviers et al, 1984; Brégeon et al, 2001). After cleavage and remethylation, both catalysed by TrmC (according to Hagervall et al, 1987), the final modification mnm5U is achieved. R can be oxygen or sulphur, corresponding to uridine or 2-thiouridine, respectively. Download figure Download PowerPoint The details of the modification steps that lead to the cmnm5U34 modification are not known. TrmE consists of three regions, an N-terminal region of about 220 amino acids, a central G domain of approximately 160 residues and a C-terminal region of 75 amino acids, which contains a motif highly conserved among the TrmE protein family. The sequence of the N-terminal region does not present homology with any known protein and could be involved in the self-assembly of the full TrmE protein (Cabedo et al, 1999). The G domain, when isolated, conserves the high intrinsic GTPase activity of the intact TrmE molecule, which suggests that removal of the N- and C-terminal regions should not substantially affect its tertiary structure (Cabedo et al, 1999). The C-terminal end contains a highly conserved CxGK motif, which resembles a CaaX box, which in case of the GTP-binding protein Ras is farnesylated in vivo and plays an important role in membrane association and cell signalling (Bourne et al, 1990). But there is no evidence for this motif to be necessary for membrane localization of TrmE. Instead, the conserved cysteine is proposed to be important in the catalysis of the modification reaction; its mutation to serine disrupts the modification of tRNA in vivo (Yim et al, 2003), which leads to the hypothesis that the first step of the modification reaction might be analogous to the C5 modification of pyrimidine catalysed by DNA cytosine-5-methyltransferase (Vilkaitis et al, 2001), where the enzyme uses a cysteine for activation of the C5 position. Studies based on the homologous 5-taurinomethyl modification of U34 in humans showed that taurine is a direct constituent of the modification (Suzuki et al, 2002). Assuming a similar TrmE-mediated modification reaction in human and bacteria (5-taurinomethyl- versus 5-cmnm-uridine) glycine instead of taurine would be incorporated in the Escherichia coli reaction. This would still leave open the question of the nature of the C1 group and the covalent bond formation with glycine. It has been shown very early that S-adenosylmethionine is not the C1 donor in charge (Hagervall et al, 1987). Mutant alleles of the GidA and TrmE homologues Mto1 and MSS1 in Saccharomyces cerevisiae reveal a respiratory-deficient phenotype (Decoster et al, 1993; Colby et al, 1998). Recent studies on GTPBP3 and Mto1, the human homologues of TrmE and GidA, lead to the suggestion that those proteins may also be involved in several human diseases like the nonsyndromic deafness or different clinical forms of myofibrillar myopathy (MERRF: myoclonic epilepsy; ragged red fibres/MELAS: mitochondrial encephalomyopathy; lactic acidosis; stroke), which are based on mutations in mitochondrial tRNA genes (Li and Guan, 2002; Li et al, 2002; Suzuki et al, 2002). Since no detailed mechanistic description of the modification reaction exists, and the role of TrmE as a regulatory or catalytic reaction partner is unclear, we decided to get insight into the reaction by solving the structure of TrmE by X-ray crystallography. Results Recombinant expression and purification TrmE from E. coli was expressed in BL21DE3 (TrmE−). The untagged protein was first purified by fractionated ammonium sulphate precipitation, followed by ion exchange chromatography on a Q-Sepharose column and size exclusion chromatography. Since crystals from E. coli TrmE did not diffract under any circumstances, we used it only for the biochemical experiments and turned to a thermophilic protein for structure determination. TrmE from Thermotoga maritima was expressed in Rosetta DE3 bacteria as an N-terminal His-tag fusion protein. It was purified by affinity chromatography via a nickel-nta-sepharose column and size exclusion chromatography using a Superdex S200 column. The construct lacking the N-terminal domain (ΔN-TrmE, G102-K454) from E. coli was expressed as an N-terminal His-tag fusion protein and purified by affinity chromatography. TrmE binds GDP/GTP with micromolar affinity During the course of structural studies, we noticed that the affinity of nucleotide was higher than anticipated from previous studies (Cabedo et al, 1999). We thus determined the equilibrium binding parameters of TrmE from E. coli by using fluorescent mant-nucleotides (mant: methylanthraniloyl). As observed for most other GTP-binding proteins, addition of protein to mant-nucleotides produces a large increase in fluorescence (Herrmann and Nassar, 1996). Using a constant concentration of nucleotide and increasing concentration of protein produces a binding isotherm (Figure 2A) that can be fitted to a binding equation. The Kd values for guanosine di- and triphosphate are in the micromolar range, with an affinity for GDP and GppNHp of 0.62 and 5.8 μM, respectively. The difference in affinity between GDP and GTP may actually be smaller since GppNHp is often found to bind with weaker affinity than GTP itself. GMP binds with an affinity lower than 100 μM, which is similar to Ras-like proteins where the affinity to GMP is orders of magnitude lower than that of GDP/GTP (Vetter and Wittinghofer, 2001). Figure 2.Nucleotide-binding properties of TrmE from E. coli. (A) Determination of the equilibrium dissociation constant Kd for mGDP and mGppNHp by fluorescence equilibrium titration. (B) Determination of the association rate constant for mGDP by stopped flow under pseudo-first-order conditions and (C) the dissociation rate constant for mGDP, as described in Materials and methods. Association and dissociation rates result in a Kd of 0.38 μM for mGDP. Download figure Download PowerPoint Additionally, kinetic parameters for mGDP binding were determined by stopped flow (Figure 2B and C). The association rate constant was measured by using pseudo-first-order conditions (TrmE in large molar excess over nucleotide). Mant-nucleotides (100 nM) were mixed with increasing concentrations of protein (1–13 μM) and the increase in mant fluorescence was monitored. The fluorescence transients were fitted single exponentially and the observed rate constants kobs were plotted against the protein concentration to give an association rate constant of 5.8 μM−1 s−1 (Figure 2B). The dissociation rate constant koff was measured by displacing mGDP from TrmE by a 200-fold excess of unlabelled nucleotide. The decrease of the fluorescence signal was fitted single exponentially for a rate constant of 2.2 s−1 (Figure 2C). The ratio of koff and kon gives the dissociation constant Kd of 0.380 μM for the TrmE to mGDP interaction, similar to the value found by equilibrium titration. Previously, an affinity for GTP in the range of 280 μM and an even lower affinity for GDP (>1 mM) were reported, which would be unusually low for a GTP-binding protein. Here we can show that the affinities are indeed much higher than reported and that the nitrocellulose filter binding method might not be appropriate for measuring affinities in the micromolar range, as has been observed before (Lenzen et al, 1998). Our measurements also indicate that the Km for GTP measured previously from the GTP dependence of the GTPase reaction (Cabedo et al, 1999) is most likely not equal to the Kd for GTP. Overall fold of TrmE Nucleotide-free TrmE from T. maritima crystallized in the space group P6(2). The crystal structure was solved at 2.3 Å using the single-wavelength anomalous dispersion (SAD) method after Se-Met incorporation. TrmE is a three-domain protein composed of the N-terminal α/β domain, residues 1–118, a central exclusively helical domain formed by residues 119–210 from the middle and the C-terminal residues 381–450, and the G-domain residues 211–380 (Figure 3B). Figure 3.Overall structure of TrmE. (A) Sequence alignment of TrmE from T. maritima (Swissprot accession number Q9WYA4), TrmE from E. coli (Swissprot accession number P25522), MSS1 from S. cerevisiae (Swissprot accession number P32559) and GTPBP3 from Homo sapiens (Swissprot accession number Q8WUW9) with secondary structure assignment determined with DSSP (Kabsch and Sander, 1983). Domains are coloured in blue (N-terminal domain), green (central helical domain) and red (G domain). Flexible regions with weak density are marked with a dashed line (switch I and II). (B) Ribbon presentation of the tertiary structure of TrmE. The N-terminal domain is shown in blue, the central helical domain in green and the G domain in red. The flexible switch regions in the G domain are indicated by dashed lines. The nucleotide-binding motifs and switch regions are marked in purple. (C) Ribbon model of the putative TrmE homodimer in two orientations. Based on the position of the second N-terminal domain (molecule B), the orientation of full-length molecule B was modelled. The homodimerization of TrmE is mainly mediated by the N-terminal domain (blue and light blue). The homodimer has an elongated shape with a size of approximately 130 Å along the longest axis and approximately 76 or 80 Å from the N-terminal domains to the G domain or from G domain to G domain, respectively. Putative nucleotide-binding sites are marked by a sphere. (D) Gel filtration of ΔN-TrmE and full-length TrmE. ΔN-TrmE elutes with a lower apparent molecular mass (62 kDa) than full-length protein (142 kDa). The equilibrium for ΔN-TrmE is on the monomer side, whereas full-length protein is present as homodimer. Download figure Download PowerPoint The crystallographic asymmetric unit contains two molecules, one of which corresponds to the full-length protein, whereas, surprisingly, the second molecule only contains the N-terminal domain, residues 1–118. Apparently, the second molecule is proteolysed in the course of the crystallization to form the observed structure. Dissolved crystals indeed showed an additional band at approximately 14 kDa corresponding to this degraded fragment (not shown). To show that TrmE is a dimer in solution also, we performed a gel filtration experiment (Figure 3D). This showed that the majority of the full-length protein runs with an apparent molecular mass of 142 kDa, with only a slight shoulder running at 65 kDa. We conclude that TrmE is most likely a dimer in solution and that the larger apparent mass is due to the elongated shape of the dimeric molecule (Figure 3C). We thus believe (see also below) that the dimerization observed in the crystal is a true representation of the dimer formed by full-length protein in solution. The full-length dimer was modelled by superimposing the full-length structure on top of the N-terminal domain (Figure 3C). The superimposition does not lead to any clashes of structural elements. The whole dimer extends over a length of 130 Å, the width and height is approximately 76 Å. In the dimer, the G domains come into proximity with the putative nucleotide-binding sites facing each other (Figure 3C). The G domain The G domain of TrmE has the canonical Ras-like fold (Figure 4A), with no insertion or deletion of secondary structural elements. The 169 residues almost match the number of residues in the minimal G domain (Vetter and Wittinghofer, 2001). TrmE contains at least four of the five conserved nucleotide-binding motifs GxxxxGKS/T or P-loop (Saraste et al, 1990), T, DxxG and NKxD (Bourne et al, 1990; 1991). The totally invariant alanine in the SAK/L (G5) motif of Ras and Gα proteins is less well conserved. The G domain in the X-ray structure is only loosely connected to the residual domains of TrmE, which may explain why the G domain alone can be expressed and exhibits a GTPase activity similar to that of the full-length protein (Cabedo et al, 1999). Figure 4.Structural details. (A) Superimposition of the G domains of TrmE and Ras (PDB 121P). The G domain of TrmE has the canonical fold of Ras without additional secondary structure elements. A total of 118 of the 169 C-α positions can be aligned with a maximal r.m.s.d. of 3.6 Å. The G domain of TrmE is shown in red and Ras in blue. (B) Superimposition of the P-loop region of TrmE and Ras. The GKS motif of TrmE is misoriented and an additional 3,10-like helix is formed, which occupies the putative position of the α- and β-phosphate of the nucleotide. Helix Gα1 is moved by 25° compared to the orientation in Ras. Colour coding is according to panel A. (C) Surface representation of TrmE calculated with GRASP (Nicholls et al, 1991), highlighting the interface between monomers. Residues forming the interface (distance <3.5 Å between molecules A and B) are coloured according to biochemical properties. Hydrophobic, acidic and basic residues involved in the interaction are shown in green, red and blue. (D) Conserved surface-exposed residues in a surface representation of TrmE. Totally conserved residues are coloured in red, 80% conserved in orange and 60% conserved in yellow (based on alignment of 79 TrmE sequences). Large conserved areas are in the nucleotide-binding region and at the dimerization interface of the N-terminal domain. Additionally, the tip of the helical domain and the region on the opposite side of the dimer interface are well conserved. Download figure Download PowerPoint Although we did not succeed in obtaining diffraction quality crystals of the nucleotide-bound form of TrmE, the nucleotide-binding site can clearly be inferred from the comparison with Ras (Figure 4A). Superimposition of the G domains of Ras and TrmE leads to a root mean square deviation (r.m.s.d.) of 1.2 Å for 70 residues, which form the central β-sheet core of the G domain. In all, 118 of the 169 C-α positions could be aligned with a maximal r.m.s.d. of 3.6 Å. The helices flanking the central β-sheet core on both sides show larger deviations. In the G1/P-loop region, which contacts the β- and γ-phosphate of the nucleotide, the r.m.s.d. is in the range of 2.7 Å. Superposition of the P-loop region of TrmE and Ras (see Figure 4B) reveals a large displacement of the GKS motif, while Gβ1 and residue G218 of TrmE still align very well with Ras. An additional 3,10-like helix is formed, which occupies the position of the α- and β-phosphate. Additionally, helix Gα1 of TrmE is moved by approximately 25°, thereby shifting the orientation of switch I and most probably also the position of Gβ2. The binding of nucleotide would break up the 3,10-like helix and allow the canonical interaction of the P-loop residues with the nucleotide and the magnesium ion. The positions of the DxxG (contacting γ-phosphate and Mg2+) and NKxD (recognition of the guanine ring) superimpose very well with Ras (r.m.s.d. <1.8 Å). In Ras-like and heterotrimeric G proteins, switch regions have been found to change their structure with the nature of the bound nucleotide. In nucleotide-free TrmE, the switch regions are highly flexible and only weak density was visible in the electron density map. In analogy to other G-domain structures, switch I and II are expected to be stably connected to the core of the G domain upon GTP binding. Since effector proteins sense the nucleotide state of GTP-binding proteins, switch I and II are involved in effector binding in regulatory G proteins. If TrmE has a regulatory role in the modification reaction, we would expect that the switch regions participate in effector binding. N-terminal domain The N-terminal domain is composed of five β-strands and three α-helices. The antiparallel β-sheet is arranged in a Greek Key motif. Helix Nα1 is inserted between Nβ2 and Nβ3. Helices Nα2 and Nα3 are located C-terminally of Nβ5, of which helix Nα3 links the N-terminal domain to the central helical domain. Molecule B in the TrmE structure corresponds to a second N-terminal domain, which forms a tight dimer with molecule A. To show that the N-terminal domain mainly mediates dimer formation, we constructed a protein that lacks the N-terminal domain (ΔN-TrmE) by deleting residues 1–101. The ΔN-TrmE construct was analysed on a gel filtration (Figure 3D) column and eluted with an apparent molecular mass of 62 kDa, much lower than that observed for the full-length protein (142 kDa). There is a slight shoulder at the position of the expected dimer, indicating that after deletion of the N-terminal domain, the monomer–dimer equilibrium is almost totally on the monomer side, while full-length protein shows the opposite behaviour. The tendency for TrmE to form higher aggregates has been shown before by gel filtration (Cabedo et al, 1999; Yamanaka et al, 2000). The dimerization interface determined from the X-ray structure spans an area of 3260 Å2, using a 1.5 Å ball to probe the surface (Figure 4C). Of this, approximately 1720 Å2 is formed by the interactions of the N-terminal domains alone. The N-terminal domain (molecule B) additionally interacts with the central helical core domain of molecule A, via an additional area of 1540 Å2. Using a model of the full-length TrmE dimer as shown in Figure 3C, the total surface would cover approximately 4800 Å2 (3260+1540 Å2), a strong indication (although no proof) for a constitutive dimer. Homodimerization involves a number of residues (Figure 4C), where the inner part of the interface is formed by more hydrophobic and the outer rim by mostly charged residues. The hydrophobic core residues are not well conserved (Figure 3A), which is not surprising since they involve a number of main-chain/main-chain or main-chain/side-chain interactions, such as between Ala45 and Arg43, and between Ala8 and Ala15/Ile16. The polar residues Lys13B, Lys90B and Asp56B stabilize the dimer formation by charged interaction with Glu131A, Glu135A and Lys146A. Central helical domain The central helical domain consists of nine α-helices. The first part with helices Hα1–Hα5 (residues 119–210) and the second part with helices Hα6–Hα9 (residues 381–450) flank the G domain on both sides. Helices Hα3, Hα4, Hα7 and Hα8 form a long four-helix bundle, which stabilizes the core structure of the domain. The domain contains a totally conserved C-terminal FCV/I/LGK motif (Figures 3A and 4D). As shown by Yim et al (2003), mutation of this totally conserved Cys447 (Cys451 in TrmE from E. coli) residue blocks the modification reaction in vivo. We find the C-terminal loop to be stabilized by interaction with helices Nα3, Hα1 and Hα7. The position of the C-terminal loop in TrmE is additionally stabilized by several highly conserved interactions. As shown in Figure 5C, the highly conserved Glu78A in the dimer interface stabilizes the conserved Arg20A, which in turn interacts with the main-chain carbonyl group of Gly449. The C-terminus is also stabilized by side-chain interaction with Thr109A. Figure 5.THF binding and catalysis. (A) Comparison of the topology of the N-terminal domain dimer with the THF-binding domain of DMGO (PDB: 1PJ7; Leys et al, 2003). Colour coding is according to the primary structure. The THF-binding site in DMGO is encoded on a single polypeptide, while homodimerization would be required to create a similar THF-binding site in TrmE. Dimerization would also create a second, symmetry-related THF-binding site. (B) The two THF-binding sites in the TrmE dimer. The binding sites are located at the periphery of the N-terminal dimerization interface. On top of the ligand, at a distance of 11 Å, the catalytic cysteine residue is located. The bound 5-formyl-THF molecules in TrmE are surrounded by an Fo−Fc electron density map contoured at 3σ. Orientation of the molecules and colour code are analogous to Figure 3C. (C) Detailed view of the THF-binding site in the apo- (left) and the cofactor-bound (right) form. The binding site for 5-formyl-THF in DMGO is shown for comparison. (D) Binding of 5-formyl-THF to TrmE determined by isothermal titration calorimetry, producing a Kd of 0.67 μM. Download figure Download PowerPoint Apart from the C-terminal loop, the only other but less well-conserved parts of TrmE are the tip region of the four-helix bundle and the back of the dimer interface (Figures 3A and 4D). Tetrahydrofolate-binding site of the N-terminal domain Using the DALI server to find homologous structures, we find a high similarity between the N-terminal domain and the tetrahydrofolate (THF)-binding domain of N,N-dimethylglycine oxidase (DMGO) (PDB: 1PJ7; z-score: 10.1; r.m.s.d.: 3.1 Å) (Leys et al, 2003). As shown in Figure 5A, the dimer of the N-terminal domains with its local noncrystallographic symmetry axis has the same topology as the THF-binding region in DMGO, but in the case of the latter, the THF-binding domain is present on a single polypeptide, whereas both N-terminal domains of TrmE would be required to form a similar THF-binding site. The homology to the THF-binding domain of DMGO suggested that TrmE might also bind THF and that some form of THF would be used to transfer a C1 group onto tRNA. To test this hypothesis, crystals of TrmE were soaked with 5-formyl-tetrahydrofolate (5-formyl-THF). The crystals diffracted up to 2.9 Å and additional density for bound 5-formyl-THF was clearly visible (Figure 5B). In DMGO, the two subdomains form a single binding site for THF, which is located at the periphery of the contact area of the subdomains. In TrmE instead, the two N-terminal domains are related to each other by a local two-fold noncrystallographic symmetry. The THF-binding site is located at the periphery of the dimer interface as seen for DMGO and involves residues from both subunits. Due to the two-fold symmetry of the N-terminal domains in TrmE, an additional THF-binding site is formed at the symmetry-related position in the TrmE dimer interface (see Figure 5A and B). Binding of 5-formyl-THF did not lead to a significant rearrangement of the backbone. The binding site for 5-formyl-THF is very similar to the one of DMGO (Leys et al, 2003) (Figure 5C). The pteridin group is bound by a double hydrogen bond to the totally invariant Glu78 from molecule A (Glu78A), which corresponds to Glu658 in DMGO. This is analogous to the double hydrogen bond between the base of guanine nucleotides and the conserved aspartic acid in the NKxD motif (Vetter and Wittinghofer, 2001). In the apo form, Glu78A stabilizes Arg20A, which contacts the C-terminal FCV/IGK loop. In the 5-formyl-THF-bound form, this interaction of Arg20A and Glu78A is broken up and the residues are pushed away from each other by the ligand. Arg20A directly stabilizes the carbonyl group of the pteridin ring but still maintains the contact to the C-terminal loop. In DMGO, a glutamate instead of an arginine indirectly binds to the carbonyl position via a bridging water molecule. The residue at position 59B (conserved as Glu, Gln, Asp or Asn) could take over the role of Asp552 in DMGO by stabilizing the N10 position of THF. Ile16B (conserved as Ile/Val), Tyr71A (Tyr/Phe) and Val61B (Val, Met, Leu, Ile) form a hydrophobic pocket for the pteridin ring comparable to Tyr651 and Leu508 in DMGO. In cells, the THF cofactor has a variable length (1–8) of glutamate residues. A number of positively charged residues are found close to the THF-binding pocket, which could stabilize a possible poly-Glu tail of THF. Although it had been suggested that THF might be a one-carbon unit (C1) donor in the modification reaction, the question as to which oxidation state of THF is used still remains. For the mechanism that is becom
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