Article Figures & Data
Figures
- FIG 1
The overall structure of the EV-D68-3Dpol. (A) The domain organization of EV-D68-3Dpol. The division of domains is based on other RdRP structures in the Picornaviridae family, and each domain is color coded as shown. Two predicated proteolysis sites are indicated by dashed vertical lines, and the corresponding residues are indicated. (B) The right-hand arrangement of the overall structure of 3Dpol. The color scheme is the same as shown in panel A. The GTP molecule and the two phenylalanines are shown as sticks. (C) The gel filtration running trace of EV-D68-3Dpol with a Superdex 200 10/300 Increase column, superimposed on the trace of four standard protein markers. The blue and red curves represent the UV absorbances, as indicated. (D) The six conserved motifs (motifs A to F) in the structure: motif A (residues 225 to 237), motif B (residues 289 to 306), motif C (residues 317 to 332), motif D (residues 336 to 353), motif E (residues 366 to 376), and motif F (residues 168 to 172). (E) The simulated Fo − Fc omit electron density map of bound GTP (contoured at 2.5σ) and its interactions with surrounding residues at the active site. The hydrogen bonds are shown by the red dashed lines (distance of ≤3.35 Å). The surrounding residues are labeled. Au, arbitrary units.
- FIG 2
The structure comparison of EVD-68-3Dpol. (A) The superposition of EV-D68-3Dpol with polio-3Dpol (green), coxsackievirus-3Dpol (cyan), HRV-16-3Dpol (deep teal), and EV71-3Dpol (magenta). The N-terminal difference (residues Lys8 to Val11) of EV-D68-3Dpol is highlighted in the dashed red oval. The four local structural differences are shown in blue and indicated by red circles. GTP is shown as spheres. (B) The superposition of the active-site residues of EV-D68-3Dpol-GTP (PDB 5XE0 ; wheat), EV71-3Dpol-GTP (PDB 3N6M ; magenta), and polio-3Dpol-GTP (PDB 1RA7 ; green). Only the residues of EV-D68-3Dpol and Lys360 from EV71-3Dpol are shown. GTP is shown in a stick model. (C) Superposition of GTP-bound EV-D68-3Dpol (PDB 5XE0 ; green) with polio-3Dpol bound by various NTPs (PDB 1RA7 in light blue; PDB 2IM2 in light magenta; PDB 2ILY in cyan) and EV71-3Dpol (PDB 3N6M in light pink). The rotation movement of the ribose ring of each NTP is indicated by the red arrow.
- FIG 3
The overall thermostability of EV-D68-3Dpol and its truncation mutant. (A) DSF measurements of the full-length EV-D68-3Dpol and the EV-D68-3Dpol-ΔN30 truncation mutant. (B) DLS measurements of the full-length EV-D68-3Dpol and the EV-D68-3Dpol-ΔN30 truncation mutant. (C) DSF measurements of four EV-D68-3Dpol mutants along with the WT. RFU, relative fluorescence units.
- FIG 4
The effects of the NTPs on the limited proteolysis profiles of EV-D68-3Dpol. (A) The digestion patterns of full-length EV-D68-3Dpol in the absence (−) or presence (+) of 10 mM NTPs (GTP, ATP, CTP, and UTP, in top-down order) by the two proteases in a 45-min time course digestion process. (B) The summary of the protective effects of GTP, ATP, CTP, and UTP, in parallel to the SDS-PAGE gel results shown on the left. The protein amount at time point 0 min was considered 100%.
- FIG 5
Comparison of the limited proteolysis patterns of EV-D68-3Dpol to those of the truncation mutant. (A) The digestion patterns of the truncation mutant and the full-length EV-D68-3Dpol. (B) Summary of the digestion of EV-D68-3Dpol-ΔN30 as well as of the full-length EV-D68-3Dpol without NTPs (graph 1), with GTP (graph 2), and with UTP (graph 3).
Tables
- TABLE 1
Diffraction data collection and refinement statistics
Parameter Value for the parametera Diffraction source Oxford Diffraction Xcalibur Nova diffractometer Wavelength (Å) 1.54 Temp (K) 100 Detector Oxford Onyx CCD Crystal detector distance (mm) 65 Rotation range per image (°) 1 Total rotation range (°) 360 Exposure time per image (s) 60 Space group P21 Cell parameters a, b, c (Å) 55.9, 81.44, 58.45 α, β, γ (°) 90, 120, 90 Resolution range (Å) 26.21–2.30 (2.42–2.30) Total no. of reflections 69,524 (9,918) No. of unique reflections 21,405 (3,156) Completeness (%) 98.1 (100) Redundancy 3.2 (3.1) I/σ(I) 6.0 (1.1) R merge b 0.201 (0.939) R rim c 0.132 (0.631) Resolution range (Å) 26.21–2.30 (2.40–2.30) No. of reflections 21,378 R work/Rfreed 0.224/0.267 No. of non-H atoms Protein 3,613 Ligand 32e Water 243 RMS deviation Bond length (Å) 0.003 Bond angle (°) 0.564 Avg B factor (Å2) Protein 35.41 Ligand 51.49e Water 32.96 Ramachandran plot (%) Favored regions 98.03 Allowed regions 1.97 - TABLE 2
DSF summary of Tm values of EV-D68-3Dpol and its mutants
Sample Mutation(s) Tm (°C) EV-D68-3Dpol 40.4 ± 0.2 EV-D68-3Dpol-ΔN30 Deletion of Gly1 to Phe30 35.4 EV-D68-3Dpol-M1 F26A F30A 37.4 ± 0.2 EV-D68-3Dpol-M2 F26D F30D 37.5 ± 0.1 EV-D68-3Dpol-M3 W399A 36.0 ± 0.2 EV-D68-3Dpol-M4 W399D 36.3 ± 0.1 - TABLE 3
Summary of total interactions of each NTP with EV-D68-3Dpol and their stability enhancement effectsa
Sample (PDB accession no.) No. of hydrogen bonds No. of salt bridges Total no. of interactions Distance to Asp234/Asp238b (Å) Stability enhancementc GTP-3Dpol-EV-D68 8 3 11 2.42 ++++ GTP-3Dpol-EV71 (3N6M ) 8 2 10 2.67 ++++ GTP-3Dpol-polio (1RA7 ) 6 3 9 2.76 ++++ UTP-3Dpol-polio (2IM2 ) 6 2 8 2.49 +++ ATP-3Dpol-polio (2ILY ) 6 3 9 2.99 ++ CTP-3Dpol-polio (2IM0 ) 5 2 7 2.83 + ↵a Cutoff distance, ≤3.35 Å.
↵b Asp234 is according to EV-D68 numbering, and Asp238 is according to EV71 numbering.
↵c Stability enhancement was measured by the unfolding temperature differences between NTP-bound EV-D68-3Dpol and apoenzyme. The number of plus signs indicates the extent of protection conferred by each NTP.
