Structure and ion-release mechanism of PIB-4-type ATPases
- Department of Biomedical Sciences, University of Copenhagen, Denmark
- Department of Chemistry, Umeå University, Sweden
- Department of Sciences, University of Copenhagen, Denmark
- Department of Laboratory Medicine, Lund University, Sweden
- Department of Chemistry and Biochemistry, The University of Texas, United States
- Cambridge Institute for Medical Research, Department of Haematology, University of Cambridge, United Kingdom
- Department of Experimental Medical Science, Lund University, Sweden
Figures
Figure 1 with 5 supplements
Overall architecture and reaction cycle.
The sCoaT structures reveal that PIB-4-ATPases comprise soluble A-, P-, and N-domains, shown in yellow, blue, and red, respectively, as well as a transmembrane domain with eight helices: MA and MB, …
Figure 1—figure supplement 1
Topology comparison.
Topological differences between PIB-ATPases and classical P-type ATPases such as sCoaT and SERCA, respectively. (a) Schematic topology of P-type ATPases showing features unique to PIB-ATPases (the …
Figure 1—figure supplement 2
Crystal packing of sCoaT E2-AlF4− compared to the E2-BeF3− crystal form of ZntA from Shigella sonnei (SsZntA, PDB ID: 4UMV).
The domains are coloured as in Figure 1b. (a) sCoaT E2-AlF4− (P21212, with one molecule per asymmetric unit). Left: view of the membrane layer. Right: 90° rotation view (compared to panel to the …
Figure 1—figure supplement 3
Electron density quality.
Final, sharpened, 2Fo-Fc electron density at σ = 1.0 (blue mesh) if not otherwise stated. The overall resolution is indicated and the structures are coloured as in Figure 1. (a) E2-BeF3− and (b) …
Figure 1—figure supplement 4
Stability of the M-domain.
Molecular dynamics (MD) simulations were performed to assess the stability of the M-domain. (a) Root mean square deviation of the M-domain in AlF4− (black), AlF4−-repeat (red), BeF3− (green), and …
Figure 1—figure supplement 5
Secondary structure stability of the M-domain.
MD simulations were performed to assess the secondary structure stability of the M-domain. Total structure (black), helix (blue), and coil (red) secondary structural elements in the (a) AlF4−, (b) …
Figure 2 with 4 supplements
Mechanistic insight into the function of PIB-4-ATPases.
(a) Functional ATPase assay in lipid–detergent solution with targeted residues in sequential order. The wild-type (WT)-specific activity using the employed experimental conditions in the presence of …
Figure 2—figure supplement 1
Metal selectivity screening and reproducibility.
(a) Screen of different transition metals, tested at 50 μM each. There is clear metal-dependent ATPase activity with Zn2+ (1.0 ± 0.01 µmol mg−1 min−1) and Cd2+ (2.8 ± 0.05 µmol mg−1 min−1), …
Figure 2—figure supplement 2
Sequence alignment of selected PIB-ATPases.
Sequence alignment of four PIB-4-ATPases, sCoaT from Sulfitobacter sp. NAS-14.1 CmCzcP from Cupriavidus metallidurans, BsZoa from Bacillus subtilis and MtCtpD from Mycobacterium tuberculosis. The PIB…
Figure 2—figure supplement 3
Comparison of E2 states overall and close views of the phosphorylation site.
The TGE loop in the E2-BeF3−-stabilized sCoaT (E2P*) is preorganized for dephosphorylation, which is not the case for SsZntA and LpCopA. (a) The overall E2P* structure of sCoaT showing the region of …
Figure 2—figure supplement 4
A-domain differences.
Superimposition of the E2-AlF4− structures of sCoaT (determined here, shown in green) and SsZntA (PDB ID 4UMW, purple) and the E2-BeF3− structure of SsZntA (PDB ID 4UMV, blue). The overall …
Figure 3
Regulation and inhibition.
(a–c) Close views of the regulatory point-of-interaction between the A- and P-domains in the E2-AlF4− structures of sCoaT, SsZntA, and SERCA (PDB IDs 4UMW and 1XP5) with the corresponding 2Fo-Fc …
Figure 4
Putative ion-release and reocclusion mechanism of PIB-4-ATPases.
Schematic model illustrating the transmembrane domain (the soluble domains have been removed for clarity) of two separate states, an E2P and an occluded E2P* conformation as the determined structure …
Tables
Table 1
Data collection and refinement statistics.
Statistics for the highest resolution shell are shown in parentheses.
| E2-BeF3− | E2-AlF4− | |
|---|---|---|
| Data collection | ||
| Wavelength (Å) | 1.0 | 1.0 |
| Space group | P 21 21 2 | P 21 21 2 |
| Cell dimensions | ||
| a, b, c (Å) | 89.0 94.5 128.8 | 89.6 93.7 128.3 |
| a, b, g (°) | 90 90 90 | 90 90 90 |
| Resolution (Å) | 47.3–3.1(3.22–3.11) | 45.6–3.3(3.37–3.25) |
| Rmerge (%) | 11.4 (276.3) | 15.5 (246) |
| I / σI | 17.8 (1.12) | 8.5 (0.98) |
| CC1/2 | 1 (0.475) | 0.99 (0.37) |
| Completeness (%) | 97.3 (99.8) | 99.2 (99.9) |
| Redundancy | 13.3 (13.8) | 6.1 (6.6) |
| Refinement | ||
| Resolution (Å) | 47.3–3.1(3.22–3.11) | 45.6–3.3(3.37–3.25) |
| No. reflections | 19,643 (1963) | 17,466 (1714) |
| Rwork / Rfree (%) | 24.4/26.8 | 21.8/25.5 |
| No. of atoms | ||
| Protein | 4,695 | 4,695 |
| Ligand/ion | 5 | 6 |
| Water | 10 | 0 |
| Average B-factors | ||
| Protein | 135.91 | 152.54 |
| Ligand/ion | 84.15 | 86.47 |
| Solvent | 79.62 | |
| R.m.s. deviations | ||
| Bond lengths (Å) | 0.004 | 0.003 |
| Bond angles (°) | 0.77 | 0.83 |
| Ramachandran statistics | ||
| Favoured (%) | 97.8 | 96.9 |
| Allowed (%) | 2.2 | 3.1 |
| Outliers (%)ClashscoreMolProbity score | 0.01.050.85 | 0.07.891.62 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Sulfitobacter sp. (strain NAS-14.1)) | NAS141_02821 | Synthetic | Uniprot: A3T2G5 | |
| Cell line (Escherichia coli) | C41(DE3) | Sigma-Aldrich | Chemically competent cells | |
| Cell line(Mycobacterium bovis) | BCG Montreal | ATCC 35735 | ||
| Software, algorithm | Phenix | RRID:SCR_014224 | https://www.phenix-online.org/ | |
| Software, algorithm | ISOLDE | https://doi.org/10.1107/S2059798318002425 | https://isolde.cimr.cam.ac.uk/ | |
| Software, algorithm | UCSF ChimeraX | RRID:SCR_015872 | https://www.cgl.ucsf.edu/chimerax/ | |
| Software, algorithm | COOT | RRID:SCR_014222 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | Pymol | RRID:SCR_000305 | http://www.pymol.org/ |
