Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub
- Section of Structural Biology, Department of Infectious Disease, Faculty of Medicine, Imperial College London, United Kingdom
- DNA:protein Interactions Unit, School of Biochemistry, University of Bristol, United Kingdom
Figures
Figure 1 with 1 supplement
RecBCD helicase activity is inhibited by gp5.9 but not Abc2.
(a) Gel-based double-stranded DNA break (DSB) resection assay. The indicated proteins or protein complexes were incubated with a linearised dsDNA substrate for the indicated times in the presence of …
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Figure 1—source data 1
Compresssed file containing images of uncropped gels and raw data associated with Figure 1.
- https://cdn.elifesciences.org/articles/83409/elife-83409-fig1-data1-v2.zip
Figure 1—figure supplement 1
Purified proteins and protein complexes used in this study.
(A) Purified RecBCD, RecBCD-Abc2P68A, and RecBCD-PPI-Abc2 complexes (1.5 µg each). The subunits are indicated. (B) Purification of gp5.9 from an insect cell extract. Lane 1, whole-cell extract; lane …
Figure 2 with 4 supplements
CryoEM structure of the RecBCD-gp5.9 complex.
(a) CryoEM map of the RecBCD-gp5.9 complex. Subunit colour coding is as follows: RecB in red, RecC in slate blue, RecD in green, and gp5.9 in yellow. (b, c) The gp5.9 dimer adopts a parallel …
Figure 2—figure supplement 1
CryoEM processing scheme for the RecBCD-gp5.9 dataset.
(a) Representative micrograph from the dataset. (b) The most populated 2D class averages generated in cryoSPARC from the cleaned 2xbinned particle set. (c) The results of cryoSPARC ab initio 3D …
Figure 2—figure supplement 2
Modelling the structure of the gp5.9 phage protein.
(a) The model of the gp5.9 dimer with the surrounding cryoEM density represented by grey mesh. (b) Superposition of the two chains of the gp5.9 model shows they share a high degree of structural …
Figure 2—figure supplement 3
Small rigid body domain movements in RecBCD facilitate binding to either DNA or gp5.9.
Both DNA and gp5.9 bind predominantly to RecBCD at the RecB arm domain and the RecC CTD. Small changes in the relative positioning of these two domains allow RecBCD to bind either DNA or gp5.9 at …
Figure 2—figure supplement 4
Rebuilding of the RecB arm domain model based on the high-resolution RecBCD-gp5.9 cryoEM map.
Side-by-side view of the density for the RecB arm in the RecBCD-gp5.9 cryoEM map, with the original RecB model (left) shown alongside the corrected RecB model (right). The large hydrophobic residues …
Figure 3
gp5.9 is a DNA mimic protein.
Charge pair interactions between R/K residues in RecBCD (blue) and either phosphates or D/E residues (red) for DNA (a), gp5.9 (b), or Gam (c). The images are all taken from the same point of view …
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Figure 3—source data 1
Complete list of interactions between the RecBCD complex and gp5.9.
- https://cdn.elifesciences.org/articles/83409/elife-83409-fig3-data1-v2.docx
Figure 4 with 6 supplements
CryoEM structures of the RecBCD-Abc2 and RecBCD-Abc2-PPI complexes.
(a) CryoEM map of the RecBCD-Abc2-DNA complex, coloured as in Figure 2 but with the DNA substrate yellow and Abc2 in orange (b) CryoEM map of the RecBCD-Abc2-PpiB-DNA complex, with PpiB in grey. (c) …
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Figure 4—source data 1
Complete list of interactions between the RecBCD complex and Abc2.
- https://cdn.elifesciences.org/articles/83409/elife-83409-fig4-data1-v2.docx
Figure 4—figure supplement 1
CryoEM processing scheme for the RecBDCD-Abc2-PPI-DNA dataset.
(a) Representative micrograph from the dataset. (b) The most populated 2D class averages generated in cryoSPARC from the cleaned 2xbinned particle set. The RELION-3 refined cryoEM maps (c) before …
Figure 4—figure supplement 2
Overview of the location of Abc2 binding in the context of the RecBCD complex.
Slabbed view of the RecBCD-Abc2-DNA structure with the Chi DNA substrate from the RecBCD Chi recognition cryoEM structure (PDB: 6SJB; Murphy, 2000) overlaid in gold with the Chi residues highlighted …
Figure 4—figure supplement 3
Abc2 binding induces minimal conformational changes in the RecBCD complex.
(a) Superposition of the RecBCD-Abc2-DNA structure (coloured as in other figures) with the corresponding RecBCD-DNA structure containing the same DNA substrate (PDB: 5LD2) in grey. The boxed panel …
Figure 4—figure supplement 4
Modelling of Abc2 and PpiB in the cryoEM maps.
(a) Density and model for the RecBCD-Abc2-DNA structure showing the Abc2 binding site on RecC. (b) Density and model for Abc2 and docked PpiB taken from the RecBCD-Abc2-PPI-DNA structure. (c) View …
Figure 4—figure supplement 5
Additional unmodelled density suggests a path for the Abc2 C-terminus breaching the RecB helicase domains.
Similar views and contouring of the blurred (a) RecBCD-Abc2-PPI-DNA and (b) RecBCD-Abc2-DNA CryoEM maps with additional, unmodelled difference density shown as a lime green surface. This suggests …
Figure 4—figure supplement 6
AlphaFold modelling of the full Abc2 protein shows a divergent C-terminus.
(a) The five top AlphaFold models for the Abc2 protein (coloured on a greyscale spectrum) are aligned to the solved Abc2 model (residues 3–66, coloured blue-to-red from the N- to the C-terminus). …
Figure 5
Hypothetical models for control of RecBCD by T7 gp5.9 and P22 Abc2.
In uninfected cells (pathway i), free DNA ends are processed by RecBCD to yield a recombinogenic 3’-terminated ssDNA overhang coated with host RecA protein. The reaction proceeds in five steps. (1) …
Tables
Table 1
Gp5.9 is a DNA mimic protein.
Ion pair contacts (<4 Å) between Arg/Lys residues in the RecBCD complex and negatively charged moieties in either DNA (phosphates) or DNA mimic proteins (Asp/Glu). Shaded rows highlight …
| RecBCD(subunit indicated) | DNA | Gp5.9 | Gam |
|---|---|---|---|
| R254 (B) | O3′-25 | E45 | D73 |
| R255 (B) | O3′-56 | D38/E39 | E118/E125 |
| K256 (B) | OP1-57/O5′-57 | E118 | |
| K264 (B) | E36 | ||
| K268 (B) | D107 | ||
| K288 (B) | OP-59 | E111 | |
| R297 (B) | OP-58 | E114 | |
| K299 (B) | OP-27 | ||
| R561 (B) | OP-69 | D4 | |
| R584 (B) | D48 | ||
| R761 (B) | O5′–68/O3′-67 | D11 | E51 |
| R822 (B) | OP-67 | D15 | |
| R823 (B) | OP-18 | ||
| R824 (B) | D21/E24 | E65/E68/E125 | |
| K828 (B) | E118 | ||
| R846 (C) | OP-10/O3′-9 | ||
| R968 (C) | OP-11/O3′-10 | ||
| R1001 (C) | OP-12 | ||
| K1066 (C) | E70/D74 | ||
| R1068 (C) | D11/D15 | ||
| K1070 (C) | E24 | E70 |
Table 2
CryoEM data collection, refinement, and validation statistics.
| gp5.9-RecBCD(EMDB-15803)(PDB 8B1R) | Abc2-RecBCD-DNA-ADPNP(EMDB-15804)(PDB 8B1T) | Abc2-RecBCD-PpiB-DNA-ADPNP(EMDB-15805)(PDB 8B1U) | |
|---|---|---|---|
| Data collection and processing | |||
| Magnification | 81,000 | 130,000 | |
| Voltage (kV) | 300 | 300 | |
| Detector | K3 | K2 | |
| Energy slit width (e–V) | 20 | 20 | |
| Electron exposure (e–/Å2) | 50 | 56 | |
| Exposure rate (e–/pixel/s) | 14.0 | 5.2 | |
| Defocus range (μm) | –1.0 to –2.5 | –1.2 to –2.4 | |
| Pixel size (Å) | 1.10 | 1.06 | |
| Movies collected | 5064 | 2500 | |
| Initial particle images (no.) | 674,546 | 185,881 | |
| Final particle images (no.) | 141,458 | 119,163 | 37,072 |
| Symmetry imposed | C1 | C1 | C1 |
| Map resolution (Å) | 3.2 | 3.4 | 3.8 |
| FSC threshold | 0.143 | 0.143 | 0.143 |
| Map resolution range (Å) | |||
| Refinement | |||
| Initial model used (PDB code) | 5MBV | 5LD2, 8B1R | 8B1T |
| Map sharpening B factor (Å2) | –50 | –50 | –50 |
| Model resolution (Å) | 3.1 | 3.3 | 3.7 |
| FSC threshold | 0.143 | 0.143 | 0.143 |
| Model to map correlation | 0.87 | 0.83 | 0.78 |
| Model composition | |||
| Non-hydrogen atoms | 21,953 | 23,986 | 24,127 |
| Protein residues | 2746 | 2869 | 2886 |
| DNA residues | - | 51 | 51 |
| Ligand molecules | 1 (Mg2+) | 1 (ADPNP) 1 (Mg2+) | 1 (ADPNP) 1 (Mg2+) |
| B factors (Å2) | |||
| Protein | 89.5 | 72.0 | 68.0 |
| DNA | - | 168.5 | 162.6 |
| Ligand | 61.0 | 50.0 | 54.5 |
| R.m.s. deviations | |||
| Bond lengths (Å) | 0.004 | 0.003 | 0.002 |
| Bond angles (°) | 0.617 | 0.493 | 0.464 |
| Validation | |||
| MolProbity score | 1.4 | 1.5 | 1.5 |
| Clashscore | 7.5 | 6.0 | 5.7 |
| Poor rotamers (%) | 0.5 | 1.4 | 1.5 |
| Ramachandran plot | |||
| Favoured (%) | 99.3 | 98.9 | 98.9 |
| Allowed (%) | 0.7 | 1.1 | 1.1 |
| Disallowed (%) | 0.0 | 0.0 | 0.0 |
