Drastic changes in conformational dynamics of the antiterminator M2-1 regulate transcription efficiency in Pneumovirinae
- Wellcome Trust Centre for Human Genetics, United Kingdom
- Diamond Light Source Ltd, United Kingdom
Figures
Figure 1 with 2 supplements
Crystal structure of HMPV M2-1.
(A) Top view (left) and side view (right) of M2-1 asymmetric tetramer in cartoon representation. Closed protomers are coloured by domain with the zinc finger in wheat, tetramerization helix in …
Figure 1—figure supplement 1
Flexibility, interfaces details and packing of M2-1 in the crystalline state.
(A) Top view (left) and side view (right) of superimposed M2-1 tetramers from two different crystals are shown in cartoon representation (apo1 and 2). (B) Overlaid closed state protomers from the …
Figure 1—figure supplement 2
Classical molecular dynamics simulations of the closed and open state models of M2-1.
(A) Model of M2-1 closed state. The N-terminal his-tag is omitted for clarity. (B) Model of M2-1 open state. (C and D) Root mean square fluctuations of each four protomers in the closed and open …
Figure 2 with 2 supplements
Solution structure of HMPV M2-1.
(A) Model for domain opening and closure. The structures are coloured by domain with the zinc finger in wheat, tetramerization domain in green, and core domain in purple. The N-terminal histag is …
Figure 2—figure supplement 1
Conformational landscape of M2-1 in the presence of nucleotides studied by SAXS.
(A and B) Effect of different buffer environments on HMPV M2-1 melting profile monitored by fluorescence-based thermal shift assay. (C–F) Two-dimensional histogram representations of radius of …
Figure 2—figure supplement 2
Model-free analysis of M2-1 conformational landscape.
(A) Normalized Kratky plots of M2-1 in the presence of 20 mM Tris pH 7.5, 150 mM NaCl, and 500 mM NDSB-201 (black), or with 3 M Gdn-HCl (pink). The grey curve is the normalized Kratky plot …
Figure 3 with 2 supplements
Interaction of the zinc finger of M2-1 with nucleic acids.
(A) Superimposition of HMPV (wheat) and RSV (split pea, PDB ID 4C3B) zinc fingers, highlighting the conservation of surface residues. (B) Structure of adenosine monophosphate bound HMPV M2-1 zinc …
Figure 3—figure supplement 1
Effect of nucleotides on HMPV M2-1 melting profile monitored by fluorescence-based thermal shift assay.
(A–D) Unfolding transitions of HMPV M2-1 at 4 µM (black spheres) in 50 mM HEPES, pH 7.5, and 150 mM NaCl and with increasing concentrations of AMP (A), UMP (B), CMP (C) or GMP (D) from 1 mM to 100 …
Figure 3—figure supplement 2
2Fo-Fc electron density maps contoured at ∼1σ, from the crystal structures of AMP and DNA bound HMPV M2-1.
(A and B) AMP molecules bound in between the zinc finger and core domains of two symmetry related protomers. (C) Binding of the DNA oligonucleotide AGTT in between the zinc finger and core domains …
Figure 4
Interaction of the core domain of M2-1 with nucleic acids.
(A) Superimposition of HMPV (purple) and RSV (lime, PDB ID 4C3D) M2-1 core domains. (B) HMPV M2-1 core domain bound to the DNA sequence AG. (C and D) Adenosine monophosphate bound core domains. The …
Figure 5 with 2 supplements
Structural characterization of M2-1/RNA complexes using SAXS.
(A) RNA induced changes on the measured SAXS profiles by addition of leader RNA (5′ACGCGAAAAAAU-3′) at 0, 1, 5, 10, 20 µM or gene end RNA (5′-AGUUAauuAAAAA-3′) at 0, 1, 5, 10, 20, 40, and 80 µM, …
Figure 5—figure supplement 1
Effect of gene end RNA (5′-AGUUAauuAAAAA-3′) on HMPV M2-1 melting profile monitored by fluorescence-based thermal shift assay.
(A) Unfolding transitions of HMPV M2-1 at 4 µM (black spheres) in 50 mM HEPES, pH 7.5, and 150 mM NaCl and with increasing concentrations of gene end RNA. (B) Variations of the protein melting …
Figure 5—figure supplement 2
Visualization of aggregated M2-1/RNA complexes by electron microscopy.
(A and B) Negatively stained M2-1/RNA complexes observed by electron microscopy, showing the formation of globular polymers with heterogeneous sizes.
Figure 6 with 1 supplement
Model of M2-1 recognition of RNA and association with the viral transcription complex.
(A) Proposed model of the recognition of the consensus gene end RNA sequence 5′-AGUUAnnnAAAAA-3′ by M2-1. The model of M2-1 in its closed state is shown in white surface representation. Residues …
Figure 6—figure supplement 1
Comparison of functionally important surfaces identified in pneumivirinae M2-1 and filoviridae VP30 core domains, based on structural alignment by secondary structure matching (shown as cartoons in A).
(B) The core domain of HMPV M2-1 is shown as a white surface, and the interacting zinc finger and tetramerization helix are shown in grey cartoon. Surfaces involved in RNA and P binding are shown in …
Tables
Table 1
X-ray data collection and refinement statistics
| Data Set | MAD–Peak | MAD–High-Energy Remote | Native | AMP-Soak | DNA-Soak |
|---|---|---|---|---|---|
| Data collection and processing statistics | |||||
| λ (Å) | 1.2828 | 1.2802 | 0.9686 | 0.9686 | 0.9795 |
| Resolution range (Å) | 33.13–2.47 | 33.13–2.51 | 44.81–2.10 | 49.80–2.01 | 63.04–2.28 |
| Space group | P21 | P21 | P21 | P21 | P21 |
| Unit cell constants (Å)/(°) | 50.2, 92.7, 82.8/90.0, 94.5, 90.0 | 50.0, 93.4, 85.2/90.0, 95.4, 90.0 | 50.1, 93.6, 85.6/90.0, 95.8, 90.0 | 50.1, 93.9, 85.5/90.0, 95.8, 90.0 | |
| Measured Reflections | 182, 655 | 167, 780 | 538, 740 | 660, 719 | 235, 027 |
| Unique Reflections | 27, 190 | 25, 798 | 45, 494 | 51, 659 | 35, 938 |
| Completeness (%) (outer shell) | 99.8 (99.9) | 99.9 (99.7) | 99.8 (99.2) | 98.8 (85.1) | 99.7 (98.8) |
| Multiplicity (%) (outer shell) | 6.7 (6.7) | 6.5 (3.3) | 11.8 (5.8) | 12.8 (5.1) | 6.5 (5.0) |
| Rpim (outer shell) | 0.039 (0.604) | 0.031 (0.367) | 0.021 (0.420) | 0.019 (0.401) | 0.023 (0.421) |
| Rmerge (outer shell) | 0.078 (1.338) | 0.062 (0.516) | 0.074 (0.937) | 0.068 (0.839) | 0.054 (0.843) |
| Mean (<I>/sd <I>) (outer shell) | 12.2 (1.7) | 14.9 (1.9) | 23.1 (2.0) | 22.5 (1.8) | 19.6 (1.8) |
| Refinement and Ramachandran statistics | |||||
| Rwork (%) | 23.34 | 19.23 | 18.64 | 19.35 | |
| Rfree (%) | 26.15 | 22.20 | 20.83 | 22.21 | |
| RMSD Bond lengths (Å) | 0.012 | 0.009 | 0.010 | 0.009 | |
| RMSD Bond angles (°) | 1.18 | 1.00 | 0.96 | 1.03 | |
| Residues in preferred regions (%) | 95.3 | 97.8 | 97.3 | 97.3 | |
| Residues in allowed regions (%) | 3.9 | 2.2 | 2.4 | 2.4 | |
| Outliers (%) | 0.8 | 0.0 | 0.3 | 0.3 | |
| PDB ID | 4CS7 | 4CS8 | 4CS9 | 4CSA | |
Table 2
SAXS-derived parameters
| Buffer conditions | c (mg/ml) | MW (kDa) | Rg (nm) | Dmax (nm) | χexp |
|---|---|---|---|---|---|
| 20 mM Tris pH 7.5 300 mM NaCl 1 M NDSB-201 | 2.00 | 93 | 4.09 | 13.71 | 0.983 |
| – | 1.50 | 89 | 3.90 | 13.22 | 0.908 |
| – | 1.00 | 82 | 3.84 | 13.01 | 0.857 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 | 0.75 | 87 | 3.52 | 12.60 | 0.839 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 + 1 M Gdn-HCl | 0.50 | 124 | 4.24 | 14.50 | 0.829 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 + 2 M Gdn-HCl | 0.50 | 140 | 4.72 | 15.34 | 0.856 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 + 3 M Gdn-HCl | 0.50 | 88 | 5.24 | 18.35 | 0.893 |
| 20 mM Tris pH 7.5 1.15 M NaCl 250 mM AMP | 1.80 | 85 | 4.30 | 14.70 | 0.986 |
| 20 mM Tris pH 7.5 150 mM NaCl 250 mM AMP | 2.00 | 97/78* | 4.01 | 13.26 | >10 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 250 mM AMP | 0.75 | 90 | 3.91 | 13.70 | 0.814 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 250 mM UMP | 0.75 | 92 | 3.86 | 13.50 | 0.843 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 250 mM CMP | 0.75 | 87 | 3.99 | 13.98 | 0.808 |
| 20 mM Tris pH 7.5 150 mM NaCl 500 mM NDSB-201 + 5 mM EDTA | 0.75 | 82 | 3.86 | 14.2 | 0.927 |
| 20 mM Tris pH 7.5 575 mM NaCl | 0.4 | 234 | 4.83 | 17.2 | N.D |
| 20 mM Tris pH 7.5 3 M NaCl | 0.4 | 463 | 8.62 | 26.7 | N.D |
| 20 mM Tris pH 7.5 1.15 M NaCl | 0.4 | 178 | 5.77 | 20.10 | N.D |
| 20 mM Tris pH 7.5 1.15 M NaCl | 0.90 | 254 | 5.91 | 20.50 | N.D |
| 20 mM Tris pH 7.5 1.15 M NaCl | 1.50 | 305 | 6.10 | 21.30 | N.D |
| 20 mM Tris pH 7.5 1.15 M NaCl | 3.00 | 580 | 7.20 | 25.20 | N.D |
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*
97 kDa assuming 90% protein + 10% RNA and 78 kDa assuming only protein.
