Article Figures & Data
Figures
- 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.
(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
- 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) 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: 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 ↵ 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/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 ↵ 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.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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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.
- Supplemental file 1
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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
