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ENZYMES AND PROTEINS

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

Rong Shi, Magda Villarroya, Rafael Ruiz-Partida, Yunge Li, Ariane Proteau, Silvia Prado, Ismaïl Moukadiri, Alfonso Benítez-Páez, Rodrigo Lomas, John Wagner, Allan Matte, Adrián Velázquez-Campoy, M.-Eugenia Armengod, Miroslaw Cygler
Rong Shi
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada
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Magda Villarroya
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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Rafael Ruiz-Partida
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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Yunge Li
2Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
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Ariane Proteau
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada
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Silvia Prado
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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Ismaïl Moukadiri
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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Alfonso Benítez-Páez
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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Rodrigo Lomas
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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John Wagner
2Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
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Allan Matte
2Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
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Adrián Velázquez-Campoy
4Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, Saragossa, Spain, and Fundación Aragón I+D (ARAID-BIFI), Diputación General de Aragón, Aragon, Spain
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M.-Eugenia Armengod
3Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler, 16-3, 46012 Valencia, Spain
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  • For correspondence: mirek@bri.nrc.ca armengod@cipf.es
Miroslaw Cygler
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada
2Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada
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  • For correspondence: mirek@bri.nrc.ca armengod@cipf.es
DOI: 10.1128/JB.00650-09
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  • FIG. 1.
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    FIG. 1.

    Crystal structure of MnmGEc. (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. MnmGEc from our structure is colored as in panel a (magenta, green, and yellow), MnmGEc with Protein Data Bank (PDB) code 3CP2 is in cyan, and MnmGCt (PDB code 3CP8) is in blue (5). (c) Superposition of the FAD-binding sites from our form of MnmGEc (magenta), MnmGEc with PDB code 3CP2 (cyan), MnmGCt (PBD code 3CP8; orange), MnmGEc(1-550) (green), and GidAr (PDB code 2CUL; gray) (3). The FAD molecules from MnmGCt and GidAr are shown in stick mode. No bound FAD molecules were found in the crystal structures of MnmGEc. Some of the residues of MnmGEc 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/ ).

  • FIG. 2.
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    FIG. 2.

    (a) Sodium dodecyl sulfate-polyacrylamide gels showing the patterns of MnmGEc 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 MnmGEc, 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) MnmGEc 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.

Tables

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  • TABLE 1.

    X-ray data and refinement statistics

    StatisticsValue(s) or data for:
    Se-MetaMnmGEcMnmGEc(1-550)
    Space group P21 P21 P3121
    Unit cell parameters
        a, b, c (Å)85.9, 144.1, 147.685.9, 144.3, 147.7144.6, 144.6, 271.0
        α, β, γ (°)90, 106.8, 9090, 106.6, 9090, 90, 120
    Wavelength (Å)0.97930.97980.9793
    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 reflections415,264480,442331,255
    No. of unique reflections133,967 (unmerged)129,26942,430
    Completeness of data (%)99.6 (99.5)97.5 (81.6)99.8 (98.5)
    R sym c 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)
    R work d (no. of reflections)0.201 (122,715)0.227 (40,204)
    R free (no. of reflections)0.238 (6,511)0.265 (2,137)
    B-factor (no. of atoms) for:
        Protein54.4 (15,648)83.3 (8,136)
        Solvent/ligand39.6 (325)79.0 (20)
    Ramachandran plot—residues in:
        Allowed regions (%)98.595.0
        Generously allowed regions (%)0.84.2
        Disallowed regions (%)0.70.8
    RMSDe
        Bond length (Å)0.0130.016
        Bond angle (°)1.52.3
    PDB code3CES3G05
    • ↵ a Se-Met, selenomethionyl (data obtained by single-wavelength anomalous diffraction).

    • ↵ b The first range is for all reflections, and the range in parentheses is for the last resolution shell.

    • ↵ c R 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.

    • ↵ d R work = (Σ F obs − F calc)/ΣF obs, where obs and calc indicate observed and calculated F values.

    • ↵ e RMSD, root mean square deviation.

  • TABLE 2.

    Properties of the MnmG mutant proteins

    Straina/plasmidbMnmG proteinMutated domaincs2U/s4U ratiodFAD protectionePutative function of mutated residue(s)f
    −ara+ara
    MG1655Wild type0.000*0.000*
    IC5241None0.0240.024
    IC5241/pBAD22None0.0260.028
    IC5241/pIC1180Flag-MnmG0.0010.000*+
    IC5241/pIC1181G13AFAD0.0150.002−FAD (Nuc) binding
    IC5241/pIC1182G15AFAD0.0150.004−FAD (Nuc) binding
    IC5241/pIC1183G13A/G15AFAD0.0240.024FAD (Nuc) binding
    IC5241/pIC1387N48A/P49AFAD0.0150.007+Catalysis or tRNA binding
    IC5241/pIC1326G52A/G53AFAD0.0250.028+Catalysis or tRNA binding
    IC5241/pIC1382K56AFAD0.0140.005+Catalysis or tRNA binding
    IC5241/pIC1327G67A/G68AFAD0.0260.020Change of local conformation
    IC5241/pIC1386K88AFAD0.0020.000*tRNA binding
    IC5241/pIC1388G89A/P90AFAD0.0160.004+Change of local conformation
    IC5241/pIC1365G156A/T157AFAD0.0230.030−FAD (Nuc) binding
    IC5241/pIC1368F158AFAD0.000*0.000*FAD (Nuc) binding
    IC5241/pIC1366R174AFAD0.0110.004−FAD (Nuc) binding
    IC5241/pIC1391R196AFAD0.0100.000*tRNA biding
    IC5241/pIC1392T199A/G200AFAD0.0210.024FAD (IAM) interaction or conformational change
    IC5241/pIC1390T201A/P202AFAD0.0200.018−FAD (IAM) binding or conformational change
    IC5241/pIC1389R204AINS0.0150.000*−FAD (IAM) binding or conformational change
    IC5241/pIC1383K283AINS0.0180.017+tRNA biding
    IC5241/pIC1384R286AINS0.0120.000*+tRNA binding
    IC5241/pIC1364Y377AFAD0.0150.013+Catalysis or tRNA binding
    IC5241/pIC1385R427AFAD0.0180.004+tRNA binding
    IC5241/pIC1442R436AFAD0.0230.018tRNA binding
    IC5241/pIC1443R440AFAD0.0190.000*tRNA binding
    IC5241/pIC1444R447AFAD0.0170.000*tRNA binding
    IC5241/pIC1440E585KC-term0.0190.004MnmE interaction?
    IC5241/pIC1480E585AC-term0.0070.000*MnmE interaction?
    IC5241/pIC1283MnmG1-535 C-term0.0240.026MnmE interaction
    • ↵ a IC5241 is MG1655 mnmG::Tn10 (8).

    • ↵ b All plasmids expressing mutant MnmG proteins are pIC1180 derivatives.

    • ↵ c FAD, 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 MnmG1-535, respectively.

    • ↵ d Levels 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.

    • ↵ f Putative 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.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Supplemental methods; Table S1, plasmids; Table 2, primers; Fig. S1, topology of EcMnmG; Fig. S2, size exclusion chromatography profiles of EcMnmG and EcMnmE; Fig. S3, HPLC chromatograms of tRNA hydrolysates and Western blot analysis of extracts from MG1655 and IC5709; Fig. S4, binding of FAD to MnmG proteins; Fig. S5, ITC analysis of MnmE binding to MnmG and MnmG1-550; Fig. S6, SPR analysis of MnmE binding to FLAG-MnmG proteins
      Word file, 2.9MB.
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Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme
Rong Shi, Magda Villarroya, Rafael Ruiz-Partida, Yunge Li, Ariane Proteau, Silvia Prado, Ismaïl Moukadiri, Alfonso Benítez-Páez, Rodrigo Lomas, John Wagner, Allan Matte, Adrián Velázquez-Campoy, M.-Eugenia Armengod, Miroslaw Cygler
Journal of Bacteriology Nov 2009, 191 (24) 7614-7619; DOI: 10.1128/JB.00650-09

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Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme
Rong Shi, Magda Villarroya, Rafael Ruiz-Partida, Yunge Li, Ariane Proteau, Silvia Prado, Ismaïl Moukadiri, Alfonso Benítez-Páez, Rodrigo Lomas, John Wagner, Allan Matte, Adrián Velázquez-Campoy, M.-Eugenia Armengod, Miroslaw Cygler
Journal of Bacteriology Nov 2009, 191 (24) 7614-7619; DOI: 10.1128/JB.00650-09
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KEYWORDS

Bacterial Proteins
Escherichia coli
Escherichia coli Proteins
RNA, Transfer

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