Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

Abstract

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

tRNA contains modified nucleosides that are posttranscriptionally generated by the activity of specific enzymes (http://modomics.genesilico.pl/sequence?seqtype=tRNA; 2). Many of these modifications frequently appear in the anticodon wobble position and are pivotal in the decoding process by stabilizing correct codon-anticodon interactions (1, 9). In Escherichia coli, the enzymes MnmE and MnmG carry out the GTP- and flavin adenine dinucleotide (FAD)-dependent incorporation of the cmnm (CH₂-NH-CH₂-COOH) group at position 5 of the wobble uridine in several tRNAs, a reaction whose specific steps remain to be elucidated (4, 7, 8). MnmG and MnmE form a heterotetrameric α₂β₂ complex in vitro (8). MnmG is a highly conserved FAD-binding protein (8) with 49% sequence identity to human MTO1.

We cloned E. coli MnmG (MnmG_Ec; NCBI gi:2367273), MnmG_Ec(1-550), and MnmE (gi:12518545) into a modified pET15b vector (Novagen). The proteins were expressed in E. coli BL21(DE3) (Novagen). Cells were grown in LB medium, induced with 100 μM isopropyl-1-thio-β-d-galactopyranoside, and incubated for ∼16 h at 16°C. Proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Mississauga, Ontario, Canada) and concentrated to ∼8 mg/ml in 20 mM Tris-HCl (pH 8.0), 0.8 M NaCl, 5% (vol/vol) glycerol, 5 mM dithiothreitol (DTT). MnmG_Ec was crystallized by the hanging-drop method, equilibrating the protein drop against reservoir solution containing 100 mM Tris-HCl (pH 7.5), 100 mM sodium formate, 6.5% (wt/vol) polyethylene glycolPEG 8000, 6% (vol/vol) ethylene glycol. The crystals belong to space group P2₁, with a = 85.9 Å, b = 144.1 Å, c = 147.6 Å, and β = 106.8°, with four molecules in the asymmetric unit (V_m = 3.03 ų Da⁻¹) (6). They differ from the previously reported crystals and diffract to significantly higher resolution (7). Selenomethionine-labeled protein [expressed in strain DL41(DE3)] crystallized under the same conditions. Crystals of MnmG_Ec(1-550) were obtained at 20°C by equilibrating protein (7 mg/ml) supplemented with the anticodon stem-loop fragment of tRNA^Glu (Dharmacon) (molar ratio, 1:1.2) against a reservoir solution of 1.3 M Li₂SO4, 0.1 M Tris-HCl (pH 8.0). They belong to space group P3₁21, with a = 144 Å.6 and c = 271.0 Å, with two molecules in the asymmetric unit (V_m = 6.39 ų Da⁻¹) and a solvent content of 80.8%.

The synchrotron data collected at the X12B beamline (NSLS, BNL) and at the SER-CAT beamline (APS, ANL) were used to solve the structures. The data were processed with HKL2000 (9). The structure of MnmG_Ec was solved at a 2.4-Å resolution by the single-wavelength anomalous diffraction method using the program SHARP/autoSHARP (2), the model was built with the help of RESOLVE (10), and the structure was refined with the program REFMAC5 (8) (see the supplemental material). The structure of MnmG_Ec(1-550) was determined by molecular replacement (MolRep [12]) and refined to a 3.5-Å resolution (Table 1). MnmG_Ec is composed of three well-ordered domains (Fig. 1a; see also Fig. S1 in the supplemental material) and a C-terminal region (residues 551 to 629) that is not visible in either crystal form (5) but is crucial for MnmE interaction (see Fig. S2 in the supplemental material). The one-dimensional nuclear magnetic resonance spectrum of MnmG_Ec suggested that the C-terminal domain is not unfolded but rather forms a molten globule or is loosely connected to the rest of MnmG_Ec.

TABLE 1.

X-ray data and refinement statistics

Statistics	Value(s) or data for:
	Se-Meta	MnmGEc	MnmGEc(1-550)
Space group	P21	P21	P3121
Unit cell parameters
a, b, c (Å)	85.9, 144.1, 147.6	85.9, 144.3, 147.7	144.6, 144.6, 271.0
α, β, γ (°)	90, 106.8, 90	90, 106.6, 90	90, 90, 120
Wavelength (Å)	0.9793	0.9798	0.9793
Resolution rangeb (Å)	50-3.00 (3.11-3.00)	50-2.41 (2.50-2.4)	50-3.49 (3.61-3.49)
No. of observed reflections	415,264	480,442	331,255
No. of unique reflections	133,967 (unmerged)	129,269	42,430
Completeness of data (%)	99.6 (99.5)	97.5 (81.6)	99.8 (98.5)
Rsymc	0.115 (0.497)	0.065 (0.356)	0.108 (0.640)
I/σ(I)	11.6 (3.0)	13.1 (2.6)	9.0 (2.2)
Rworkd (no. of reflections)		0.201 (122,715)	0.227 (40,204)
Rfree (no. of reflections)		0.238 (6,511)	0.265 (2,137)
B-factor (no. of atoms) for:
Protein		54.4 (15,648)	83.3 (8,136)
Solvent/ligand		39.6 (325)	79.0 (20)
Ramachandran plot—residues in:
Allowed regions (%)		98.5	95.0
Generously allowed regions (%)		0.8	4.2
Disallowed regions (%)		0.7	0.8
RMSDe
Bond length (Å)		0.013	0.016
Bond angle (°)		1.5	2.3
PDB code		3CES	3G05

^aSe-Met, selenomethionyl (data obtained by single-wavelength anomalous diffraction).

^bThe first range is for all reflections, and the range in parentheses is for the last resolution shell.

^cR_sym = (Σ I_obs − I_avg)/ΣI_avg, where obs and avg indicate observed and average values of I. Values in parentheses are R_sym for the last resolution shell.

^dR_work = (Σ F_obs − F_calc)/ΣF_obs, where obs and calc indicate observed and calculated F values.

^eRMSD, root mean square deviation.

FIG. 1.

Crystal structure of MnmG_Ec. (a) MnmG monomer. The N-terminal FAD-binding domain is colored magenta, the insertion domain is green, and the α-helical domain is yellow. The locations of disordered loops (residues 167 to 179 and 263 to 277) are schematically marked by dotted lines. Arrows indicate a deep cleft between the FAD-binding and insertion domains. (b) Stereoview looking down at the cleft between the insertion and FAD-binding domains. The insertion domain is at the top, and the FAD-binding domain is at the bottom. Several MnmG molecules are superimposed, showing conformational flexibility of this putative tRNA-binding cleft. MnmG_Ec from our structure is colored as in panel a (magenta, green, and yellow), MnmG_Ec with Protein Data Bank (PDB) code 3CP2 is in cyan, and MnmG_Ct (PDB code 3CP8) is in blue (5). (c) Superposition of the FAD-binding sites from our form of MnmG_Ec (magenta), MnmG_Ec with PDB code 3CP2 (cyan), MnmG_Ct (PBD code 3CP8; orange), MnmG_Ec(1-550) (green), and GidA_r (PDB code 2CUL; gray) (3). The FAD molecules from MnmG_Ct and GidA_r are shown in stick mode. No bound FAD molecules were found in the crystal structures of MnmG_Ec. Some of the residues of MnmG_Ec discussed in the text are shown explicitly. The major structural deviation in this region is limited to the loop ¹⁵³LTVGTFLDG¹⁶¹. This figure was prepared with PyMOL (http://pymol.org/).

MnmG_Ec and MnmG_Ec(1-550) form dimers in the crystals and in solution. The N-terminal, FAD-binding domain consists of segments comprising residues 1 to 201 and 342 to 458. Residues 202 to 341 form an insertion domain. Residues 459 to 550 form an α-helical domain, protruding outward from the N-terminal domain (Fig. 1a).

Comparison of the available MnmG structures identified three highly mobile segments that might be functionally important (loops encompassing approximately residues 165 to 180 and 245 to 295 and the region consisting of approximately residues 420 to 435 [MnmG_Ec numbering]) (Fig. 1b). The segment of residues 155 to 160, adjoining the first loop and partially disordered in various structures, forms part of the FAD's adenosine-binding site, and its conformation varies among MnmGs (Fig. 1c). All conformations observed in MnmG_Ecs are incompatible with FAD binding (Fig. 1c). The intrinsic flexibility of this region likely contributes to the rather low affinity of MnmG_Ec for FAD (see below), with a K_d (dissociation constant) of 3.0 ± 0.4 μM as measured by isothermal titration calorimetry (ITC) (see Fig. S3 in the supplemental material). The second segment, Thr245 to Ile295, is also disordered to a different extent in various MnmGs and adopts different conformations in ordered parts (Fig. 1b). The largest deviation occurs for residues D²⁸⁹RNQH²⁹³ at the top of this segment and residues Thr247 to Asn248 in the next β-strand. The region from Thr419 to Leu437 (Fig. 1b) is slightly longer in Chlorobium tepidum MnmG (MnmG_Ct), and the conformation of this region differs from that in MnmG_Ec. Specifically, the N-terminal end of MnmG_Ct lies close to the FAD molecule, with Arg423 contacting the flavin rings. The analogous Arg427 in MnmG_Ec is within sequence T⁴²²KEPYR⁴²⁷MFT⁴³⁰, identical to the corresponding sequence in MnmG_Ct and highly conserved among MnmGs (6). However, Arg427 is directed toward the solvent, over 13 Å away from its counterpart in MnmG_Ct. This structural flexibility is likely biologically relevant, providing a partner-induced fit mechanism.

MnmG_Ec is an FAD-binding protein with the tendency to lose its FAD cofactor (7, 14). In fact, it has not been possible to obtain crystals of MnmG_Ec with FAD, as far as we know. Here, the binding of FAD to MnmG_Ec was determined by ITC in a Microcal VP-ITC calorimeter (MicroCal, Northampton, MA). Experiments consisted of the titration of a 225 μM solution of nucleotide to a 15 μM MnmG solution at 25°C in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM MgCl₂. Binding was observed for FAD with an affinity of 3.0 ± 0.4 μM (see Fig. S4 in the supplemental material).

Based on findings from structural comparisons, we mutated highly conserved MnmG residues to alanines and explored the abilities of the resulting proteins to modify tRNA in vivo and to be protected by FAD against trypsinolysis in vitro. We analyzed the nucleoside compositions of tRNAs extracted from strain IC5241 (mnmG::Tn10) and IC5241 carrying pIC1180 or its derivative plasmids (see Table S1 in the supplemental material), which express, under the control of P_BAD, wild-type or mutant MnmG protein. To this end, strains were grown in LB until the optical density at 600 nm reached 0.4. The culture was then divided into two equal parts, arabinose (0.2%) was added to one of them, and incubation was continued until the optical density at 600 nm reached 1.0. Cells were subsequently collected and processed as described previously for nucleoside analysis (5). In the absence of MnmG, the accumulation of s²U (defective conversion of wobble s²U into mnm⁵s²U) was observed (Table 2; see also Fig. S4a in the supplemental material), whereas expression of Flag-MnmG (wild type) reduced the fraction of s²U to a level close to that observed in a wild-type strain, even in the absence of an inducer (arabinose), indicating a leakiness of the pIC1180 expression system. In fact, wild-type levels of Flag-MnmG were observed without the inducer (see Fig. S4b in the supplemental material). The mutation of most of the selected residues impaired the tRNA-modifying function of MnmG (as summarized in Table 2). In some cases, the phenotype was rescued by overexpression of the mutant protein.

TABLE 2.

Properties of the MnmG mutant proteins

Straina/plasmidb	MnmG protein	Mutated domainc	s2U/s4U ratiod		FAD protectione	Putative function of mutated residue(s)f
			−ara	+ara
MG1655	Wild type		0.000*	0.000*
IC5241	None		0.024	0.024
IC5241/pBAD22	None		0.026	0.028
IC5241/pIC1180	Flag-MnmG		0.001	0.000*	+
IC5241/pIC1181	G13A	FAD	0.015	0.002	−	FAD (Nuc) binding
IC5241/pIC1182	G15A	FAD	0.015	0.004	−	FAD (Nuc) binding
IC5241/pIC1183	G13A/G15A	FAD	0.024	0.024		FAD (Nuc) binding
IC5241/pIC1387	N48A/P49A	FAD	0.015	0.007	+	Catalysis or tRNA binding
IC5241/pIC1326	G52A/G53A	FAD	0.025	0.028	+	Catalysis or tRNA binding
IC5241/pIC1382	K56A	FAD	0.014	0.005	+	Catalysis or tRNA binding
IC5241/pIC1327	G67A/G68A	FAD	0.026	0.020		Change of local conformation
IC5241/pIC1386	K88A	FAD	0.002	0.000*		tRNA binding
IC5241/pIC1388	G89A/P90A	FAD	0.016	0.004	+	Change of local conformation
IC5241/pIC1365	G156A/T157A	FAD	0.023	0.030	−	FAD (Nuc) binding
IC5241/pIC1368	F158A	FAD	0.000*	0.000*		FAD (Nuc) binding
IC5241/pIC1366	R174A	FAD	0.011	0.004	−	FAD (Nuc) binding
IC5241/pIC1391	R196A	FAD	0.010	0.000*		tRNA biding
IC5241/pIC1392	T199A/G200A	FAD	0.021	0.024		FAD (IAM) interaction or conformational change
IC5241/pIC1390	T201A/P202A	FAD	0.020	0.018	−	FAD (IAM) binding or conformational change
IC5241/pIC1389	R204A	INS	0.015	0.000*	−	FAD (IAM) binding or conformational change
IC5241/pIC1383	K283A	INS	0.018	0.017	+	tRNA biding
IC5241/pIC1384	R286A	INS	0.012	0.000*	+	tRNA binding
IC5241/pIC1364	Y377A	FAD	0.015	0.013	+	Catalysis or tRNA binding
IC5241/pIC1385	R427A	FAD	0.018	0.004	+	tRNA binding
IC5241/pIC1442	R436A	FAD	0.023	0.018		tRNA binding
IC5241/pIC1443	R440A	FAD	0.019	0.000*		tRNA binding
IC5241/pIC1444	R447A	FAD	0.017	0.000*		tRNA binding
IC5241/pIC1440	E585K	C-term	0.019	0.004		MnmE interaction?
IC5241/pIC1480	E585A	C-term	0.007	0.000*		MnmE interaction?
IC5241/pIC1283	MnmG1-535	C-term	0.024	0.026		MnmE interaction

^aIC5241 is MG1655 mnmG::Tn10 (8).

^bAll plasmids expressing mutant MnmG proteins are pIC1180 derivatives.

^cFAD, FAD-binding domain; INS, insertion domain (equivalent to I2 domain described in reference 5). The C-terminal region (C-term) of MnmG carries a point mutation and is deleted in the E585K mutant and MnmG_1-535, respectively.

^dLevels of nucleoside s²U detected in the high-performance liquid chromatography analysis of tRNA purified from the indicated strain. −ara and +ara indicate the absence and presence of the inducer arabinose in the growth medium. The numbers are calculated as the absorbance of s²U relative to the absorbance of s⁴U at 314 nm and are the means of results from at least two independent experiments. The asterisks indicate that s²U was undetectable.

^e+ or − indicates that the protein is protected or not protected by the cofactor against trypsinolysis.

^fPutative function assigned to the residue(s) on the basis of structural, tRNA modification, and proteolysis data. Nuc, nucleotide moiety of FAD; IAM, isoalloxazine moiety of FAD.

Limited proteolysis was used to explore the role of MnmG residues in FAD binding. FLAG-MnmG proteins (5 μg) were incubated with the indicated nucleotide concentrations in 25 μl of digestion buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM MgCl₂, 5 mM DTT) for 2 h at room temperature. Proteolysis reactions were started by adding TPCK (tosylsulfonyl phenylalanyl chloromethyl ketone)-trypsin (Sigma) at an MnmG-trypsin ratio of 300:1 (wt/wt) at 30°C. After 10 min incubation, reactions were stopped with the addition of phenylmethylsulfonyl fluoride (2 mM final concentration) and 4× Laemmli sample buffer, and the products were heated for 8 min at 95°C and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FAD protected MnmG_Ec from trypsinolysis, suggesting binding-induced conformational changes (Fig. 2a). The mutation of residues located in the vicinity of FAD (G13A, G15A, G156A/T157A, R174A, T201A/P202A, and R204A) impaired the protection (Fig. 1c and 2b), supporting evidence for the interaction of these residues with FAD or their role in the FAD-induced conformational change. These mutations also affect the tRNA modification function of MnmG (Table 2), in agreement with the idea that this function is FAD dependent (8). Mutation F158A does not impair the tRNA modification (Table 2), indicating that the aromatic ring of phenylalanine 158 is not critical for FAD binding.

FIG. 2.

(a) Sodium dodecyl sulfate-polyacrylamide gels showing the patterns of MnmG_Ec digestion by limited trypsinolysis in the presence of increasing concentrations (0 to 1 mM) of FAD and NADPH (negative control). Proteins were visualized by Coomassie staining. Positions of mass markers and their sizes, in kilodaltons, are indicated to the left. Full-length MnmG_Ec, at the top of each gel, migrates as a protein of 70 kDa. Bands below the full-length protein result from proteolysis. −, absent; +, present. (b) Sodium dodecyl sulfate-polyacrylamide gels showing the patterns of digestion of wild-type (WT) MnmG_Ec and various mutants (indicated by the corresponding mutations) by limited trypsinolysis in the presence of increasing concentrations (0 to 5 mM) of FAD. Molecular mass markers are shown as described above.

Residues Ser46 to Pro49, Gly52 to Gly53, Lys56, Ile343, Thr375, and Tyr377 are presumed to be in the vicinity of the FAD isoalloxazine ring. However, N48A/P49A, G52A/G53A, K56A, and Y377A mutants, while displaying partial or complete loss of function, are protected by FAD against trypsinolysis, which suggests that the roles of the mutated residues in FAD binding are minimal and that these residues are involved instead in maintaining the active-site architecture.

A double mutation, G67A/G68A, leads to a complete loss of function. Although according to our structural model, Gly67 and Gly68 are not directly involved in FAD binding, they are located at the turn between two α-helices involved in FAD binding and affect the local structure.

A distinguishing feature of MnmG proteins is a large cleft formed between the FAD-binding domain and the insertion domain (Fig. 1b). The cleft is lined with a series of conserved basic residues, including Lys56, Arg82, Lys88, Arg96, Lys198, Arg427, and Arg432, suitable for interaction with a negatively charged tRNA molecule. These basic residues are situated in the FAD-binding domain on one side of the cleft, while Lys283, Arg286, and Arg290 from the insertion domain are on the opposite side of the cleft. The presence of this cluster of basic residues gives the cleft a highly positive electrostatic potential. The FAD cofactor-binding pocket is located just below this cleft, with the riboflavin moiety partially exposed to solvent within the cleft. Interestingly, some of the above-mentioned residues are located in the mobile loops consisting of approximately residues 245 to 295 and 420 to 435 within the cleft. Mutations K56A, K283A, R286A, and R427A, while affecting tRNA modification, do not impair the FAD-induced protection against trypsinolysis (Table 2 and Fig. 2b), which is in line with a putative role of the corresponding residues in tRNA, but not FAD, binding. ITC measurements (see Fig. S3 in the supplemental material) confirmed that K283A has no effect on FAD binding (K_d for the K283A mutant, 3.7 ± 0.5 μM) and that G13A (affecting the first Gly residue of the consensus dinucleotide binding motif GXGXXG) reduces the affinity by 10-fold (K_d for the G13A mutant, 27 ± 4 μM). The ITC data for both mutants are in agreement with the results obtained from the proteolysis analysis, supporting the validity of this approach to gain information on the capabilities of MnmG mutants to bind FAD or undergo conformational changes associated with FAD binding. Note that other mutations affecting basic residues neighboring the mobile region from approximately residue 420 to residue 435 (R436A, R440A, and R447A) also impair the tRNA modification function, whereas the K88A mutant exhibits a nearly wild-type phenotype (Table 2).

Mutation G89A/P90A causes partial loss of the tRNA modification function, but it does not affect the FAD-induced resistance to trypsinolysis (Table 2 and Fig. 2b), suggesting that the corresponding residues, while affecting activity, are not associated with binding to the cofactor. Gly89 and Pro90 are situated in the turn between β4 and β5 (Fig. 1a; see also Fig. S1 in the supplemental material), and their change to alanine may affect the putative tRNA-binding site.

ITC and surface plasmon resonance experiments were conducted to quantify the binding of MnmE_Ec to MnmG_Ec (see Fig. S5a and S6 in the supplemental material). In the ITC experiments, a 150 μM solution of MnmE was titrated into a 10 μM solution of the MnmG or MnmG_1-550 protein. Surface plasmon resonance studies were carried out at 25°C with a Biacore T100 instrument (Biacore AB, Uppsala, Sweden). Anti-FLAG M2 monoclonal antibody from Sigma (100 ng/μl in 10 mM sodium acetate, pH 4.5) was immobilized onto a CM-5 sensor chip (Biacore AB) at 10,000 to 12,000 resonance units using an amine coupling kit (Biacore AB). FLAG-MnmG at 1 μM in TBS buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl₂, 0.005% Tween 20) was immobilized by capture, giving about 1,500 relative units. MnmE in TBS buffer at various concentrations was passed over the sensor chip at a flow rate of 30 μl/min, and the interactions were monitored for 1 min. The sensor surface was washed with TBS buffer to detect dissociation and then regenerated with a pulse of 5 mM NaOH (15 s at 60 μl/min). T100 Biacore evaluation software (Biacore AB) was used to evaluate the data. We obtained a K_d value of 3.3 μM by ITC and a value of 2.0 μM by SPR (see Fig. S5a and S6 in the supplemental material). In agreement with results from size exclusion chromatography (see Fig. S2 in the supplemental material), no binding of MnmE_Ec to MnmG_Ec(1-550) was observed by using ITC (see Fig. S5b in the supplemental material) and surface plasmon resonance. The expression of MnmG_Ec(1-550) and MnmG_Ec(1-535) in appropriate mnmG::Tn10 strains was unable to restore the wild-type phenotype (Table 2 and data not shown), suggesting that the formation of the MnmE-MnmG complex mediated by the MnmG C-terminal region is crucial for tRNA modification. Curiously, this function was impaired by mutation E585K and, to a lesser extent, E585A (Table 2), but neither of these changes affected the MnmG-MnmE interaction (see Fig. S6b in the supplemental material), in contrast to the data in a previous report (5). Thus, the role of Glu585 remains to be determined.