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Literature summary for 7.4.2.8 extracted from
- X-ray crystal structures of the type IVb secretion system DotB ATPases (2018), Protein Sci., 27, 1464-1475 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expressed in Escherichia coli C43 cells | Legionella pneumophila |
| gene dotB, recombinant expression of Strep-tagged enzyme in Escherichia coli strain C43 | Legionella pneumophila |
| gene YpsIP31758_B0110, plasmid-encoded enzyme, recombinant expression in Escherichia coli strain C43 | Yersinia pseudotuberculosis |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| purified recombinant Strep-tagged enzyme, sitting drop vapor diffusion technique, by mixing 200 nl of 5 mg/ml protein in 50 mM Tris, 0.2 M NaCl, 5% glycerol, 1 mM MgCl2, and 1 mM AMP-PNP with reservoir solution containing 0.1 M Na/cacodylate, pH 5.5, and 12% PEG 8000, equilibration against the reservoir solution, overnight at 16°C, X-ray diffraction structure determination and analysis at 2.75 A resolution, molecular replacement and modelling using the structure of the PilT (PDB 2EWV) C-terminal domain and PilT2 (PDB 5FL3) N-terminal domain as search templates | Yersinia pseudotuberculosis |
| purified recombinant Strep-tagged enzyme, sitting drop vapor diffusion technique, by mixing 200 nl of 5 mg/ml protein in 50 mM Tris, 0.2 M NaCl, 5% glycerol, 1 mM MgCl2, and 1 mM AMP-PNP with reservoir solution containing 1.2 M of Na/K phosphate, pH 7.2, equilibration against the reservoir solution, overnight at 16°C, X-ray diffraction structure determination and analysis at 3.19 A resolution, molecular replacement and modelling using the structure of DotBY from Yersinia pseudotuberculosis as search model | Legionella pneumophila |
| sitting drop vapor diffusion method, using 1.2 M Na/K phosphate buffer pH 7.2 for subunit DotBL or 0.1M Na/cacodylate buffer pH5.5 and 12% (w/v) PEG 8000 for subunit DotBY | Legionella pneumophila |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C110R | naturally occurring mutation, a class I mutant, has hexamerization defects, and is located at the NTDn/CTDn11 interface | Legionella pneumophila |
| D35N | naturally occurring mutation, a class IV mutant, has hexamerization defects, and is located at the NTDn/CTDn11 interface | Legionella pneumophila |
| G234D | naturally occurring mutation, a class IV mutant | Legionella pneumophila |
| H253R | naturally occurring mutation, a class IV mutant | Legionella pneumophila |
| K162N/Q | naturally occurring mutation, a class I mutant | Legionella pneumophila |
| K162Q | inactive | Legionella pneumophila |
| K182E | naturally occurring mutation, a class I mutant, has hexamerization defects, and is located at the NTDn/CTDn11 interface | Legionella pneumophila |
| L111P | naturally occurring mutation, a class I mutant, has hexamerization defects, and is located at the NTDn/CTDn11 interface | Legionella pneumophila |
| N180I | naturally occurring mutation, a class IV mutant, has hexamerization defects, and is located at the NTDn/CTDn11 interface | Legionella pneumophila |
| R104E | inactive | Legionella pneumophila |
| R104E | naturally occurring mutation, a class IV mutant, located at the NTDn/CTDn11 interface | Legionella pneumophila |
| R123E | inactive | Legionella pneumophila |
| R123E | naturally occurring mutation, a class IV mutant, located at the NTDn/CTDn11 interface | Legionella pneumophila |
| R270C | naturally occurring mutation, the mutant is partially functional and can harbor a loss of function due a conformation-switch defect, since Arg270 is part of the critical lower CTDn/CTDn11 interface | Legionella pneumophila |
| S163L | naturally occurring mutation, a class I mutant | Legionella pneumophila |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.99 | - |
ATP | pH 8.0, 22°C, recombinant enzyme | Legionella pneumophila |  |
| 0.99 | - |
ATP | subunit DotBL, at 25°C, pH not specified in the publication | Legionella pneumophila | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Legionella pneumophila | |
| Mg2+ | required | Yersinia pseudotuberculosis | |
| Mg2+ | 2 mM used in assay conditions | Legionella pneumophila | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 256700 | - |
SEC-MALS | Legionella pneumophila |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | Legionella pneumophila | - |
ADP + phosphate | - |
? | |
| ATP + H2O | Yersinia pseudotuberculosis | - |
ADP + phosphate | - |
? | |
| ATP + H2O | Yersinia pseudotuberculosis IP31758 | - |
ADP + phosphate | - |
? | |
| ATP + H2O + cellular protein[side 1] | Legionella pneumophila | - |
ADP + phosphate + cellular protein[side 2] | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Legionella pneumophila | O52185 | - |
- |
| Yersinia pseudotuberculosis | A0A0U1QTI9 | serotype O:1b | - |
| Yersinia pseudotuberculosis IP31758 | A0A0U1QTI9 | serotype O:1b | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant Strep-tagged enzyme from Escherichia coli strain C43 by solubilization with C3-Emulsiflex, affinity chromatography, ultrafiltration, and gel filtration | Legionella pneumophila |
| recombinant Strep-tagged enzyme from Escherichia coli strain C43 by solubilization with C3-Emulsiflex, affinity chromatography, ultrafiltration, and gel filtration | Yersinia pseudotuberculosis |
| StrepTrap column chromatography and Superdex 200 gel filtration | Legionella pneumophila |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | - |
Legionella pneumophila | ADP + phosphate | - |
? | |
| ATP + H2O | - |
Yersinia pseudotuberculosis | ADP + phosphate | - |
? | |
| ATP + H2O | - |
Yersinia pseudotuberculosis IP31758 | ADP + phosphate | - |
? | |
| ATP + H2O + cellular protein[side 1] | - |
Legionella pneumophila | ADP + phosphate + cellular protein[side 2] | - |
? | |
| additional information | phosphate detection by malachite green assay | Legionella pneumophila | ? | - |
- |
|
| additional information | phosphate detection by malachite green assay | Yersinia pseudotuberculosis | ? | - |
- |
|
| additional information | phosphate detection by malachite green assay | Yersinia pseudotuberculosis IP31758 | ? | - |
- |
|
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| hexamer | 6 * 44000, SDS-PAGE, DotBL forms an asymmetric hexamer, subunit interaction analysis, three intersubunit interfaces, protein-protein interfaces between subunits, detailed overview | Legionella pneumophila |
| homohexamer | 6 * 44000, SDS-PAGE | Legionella pneumophila |
| More | the DotBL subunit is composed of an about 150 amino acids long N-terminal domain (NTD), comprising 6 beta-strands and 3 alpha-helices, and an about 250 amino acids C-terminal domain (CTD) consisting of 7 beta-strands and 10 alpha-helices. The two domains are connected by a 10 amino acids proline-rich linker | Legionella pneumophila |
| More | the DotBY subunit is composed of an about 150 amino acids long N-terminal domain (NTD), comprising 6 beta-strands and 3 alpha-helices, and an about 250 amino acids C-terminal domain (CTD) consisting of 7 beta-strands and 10 alpha-helices. The two domains are connected by a 10 aminoacids proline-rich linker | Yersinia pseudotuberculosis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| ATPase | - |
Legionella pneumophila |
| ATPase | - |
Yersinia pseudotuberculosis |
| DotB | - |
Legionella pneumophila |
| DotB ATPase | - |
Legionella pneumophila |
| DotB ATPase | - |
Yersinia pseudotuberculosis |
| DotBL | - |
Legionella pneumophila |
| DotBL | subunit | Legionella pneumophila |
| DotBY | - |
Yersinia pseudotuberculosis |
| DotBY | subunit | Legionella pneumophila |
| type IV secretion system protein DotB | UniProt | Yersinia pseudotuberculosis |
| type IVb secretion system ATPase | - |
Legionella pneumophila |
| type IVb secretion system ATPase | - |
Yersinia pseudotuberculosis |
| type IVb secretion system DotB ATPase | - |
Legionella pneumophila |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 22 | - |
assay at room temperature | Legionella pneumophila |
| 22 | - |
assay at room temperature | Yersinia pseudotuberculosis |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 674 | - |
ATP | pH 8.0, 22°C, recombinant enzyme | Legionella pneumophila | |
| 674 | - |
ATP | subunit DotBL, at 25°C, pH not specified in the publication | Legionella pneumophila | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
about, assay at | Legionella pneumophila |
| 8 | - |
about, assay at | Yersinia pseudotuberculosis |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | ATPase DotB belongs to the VirB11 family of proteins, but sequence analysis reveals that DotB might be more related to PilT/EpsE family proteins than VirB11 family proteins. The subunits DotBL from Legionella pneumophila and DotBY from Yersinia pseudotuberculosis display a very similar topology | Yersinia pseudotuberculosis |
| evolution | ATPase DotB belongs to the VirB11 family of proteins, but sequence analysis reveals that DotB might be more related to PilT/EpsE family proteins than VirB11 family proteins. The subunits DotBL from Legionella pneumophila and DotBY from Yersinia pseudotuberculosis display a very similar topology. The N-terminal domain of DotBL adopts a PAS-like fold, similar to PilT, EpsE, and HP0525 | Legionella pneumophila |
| additional information | enzyme structure determination, analysis, and comparison, overview | Legionella pneumophila |
| additional information | enzyme structure determination, analysis, and comparison, overview | Yersinia pseudotuberculosis |
| physiological function | Legionella pneumophila utilizes a type IVb secretion (T4bS) system termed dot/icm to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA1 ATPase, a protein called DotB | Legionella pneumophila |
| physiological function | the type IVb secretion (T4bS) system termed dot/icm secretes protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA1 ATPase, a protein called DotB | Yersinia pseudotuberculosis |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 680.8 | - |
ATP | pH 8.0, 22°C, recombinant enzyme | Legionella pneumophila | |
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