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Literature summary for 7.4.2.8 extracted from
- Structural and biochemical characterization of Spa47 provides mechanistic insight into type III secretion system ATPase activation and Shigella virulence regulation (2016), J. Biol. Chem., 291, 25837-25852 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expressed in Escherichia coli Tuner(DE3) cells | Shigella flexneri |
| gene spa47, sequence comparisons | Shigella flexneri |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| Shigella T3SS ATPase Spa47 mutant Spa47DELTA1-79, crystal structure analysis. Crystallization of purified enzyme mutants K165A, E188A, and R350A, X-ray diffraction structure determination and analysis at 1.8-2.7 A resolution | Shigella flexneri |
| vapor diffusion method, using 0.1 M Tris, pH 8.5, 0.2 M ammonium acetate, 0.2 M lithium sulfate, 20-26% (w/v) PEG 4000, and 4.5-9.5% (v/v) (+/-)-2-methyl-2,4-pentanediol | Shigella flexneri |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| E188A | inactive | Shigella flexneri |
| E188A | site-directed mutagenesis, ATPase-inactive active site mutant, the mutation has little effect on the global structure of the protein | Shigella flexneri |
| K165A | inactive | Shigella flexneri |
| K165A | site-directed mutagenesis, ATPase-inactive active site mutant, the mutation has little effect on the global structure of the protein | Shigella flexneri |
| additional information | N-terminal domain truncation results in strictly monomeric enzyme that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues | Shigella flexneri |
| additional information | although N-terminal domain truncation is necessary for crystal formation, it results in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. ATPase inactive full-length Spa47 point mutants show, that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. Generation of an activated hexameric Spa47 model. Construction and expression of diverse ATPase-inactive Spa47 mutants in Shigella, phenotypes, overview. The truncated mutant Spa47DELTA1-79 is ATPase-inactive | Shigella flexneri |
| R350A | inactive | Shigella flexneri |
| R350A | site-directed mutagenesis, ATPase-inactive active site mutant, the mutation has little effect on the global structure of the protein | Shigella flexneri |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| membrane | - |
Shigella flexneri | 16020 | - |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | Shigella flexneri | - |
ADP + phosphate | - |
? |  |
| ATP + H2O + cellular protein[side 1] | Shigella flexneri | - |
ADP + phosphate + cellular protein[side 2] | - |
? | |
| ATP + H2O + cellular protein[side 1] | Shigella flexneri 2457T | - |
ADP + phosphate + cellular protein[side 2] | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Shigella flexneri | P0A1C1 | serotype 2a | - |
| Shigella flexneri | Q6XVW8 | - |
- |
| Shigella flexneri 2457T | P0A1C1 | serotype 2a | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| Q Sepharose column chromatography and Superdex 200 gel filtration | Shigella flexneri |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O | - |
Shigella flexneri | ADP + phosphate | - |
? | |
| ATP + H2O + cellular protein[side 1] | - |
Shigella flexneri | ADP + phosphate + cellular protein[side 2] | - |
? | |
| ATP + H2O + cellular protein[side 1] | - |
Shigella flexneri 2457T | ADP + phosphate + cellular protein[side 2] | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| hexamer | structure overview | Shigella flexneri |
| hexamer | 6 * 38900, calculated from amino acid sequence | Shigella flexneri |
| More | analysis of Spa47 oligomeric distribution | Shigella flexneri |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| ATPase | - |
Shigella flexneri |
| Spa47 | - |
Shigella flexneri |
| T3SS ATPase | - |
Shigella flexneri |
| type III secretion system ATPase | - |
Shigella flexneri |
General Information
| General Information | Comment | Organism |
|---|---|---|
| malfunction | complementing a spa47 null Shigella flexneri strain with the inactive Spa47K165A mutant results in the same lack of invasiveness seen for the null strain. The hemolysis results presented suggest that Shigella strains lacking the gene for Spa47 or strains complemented with ATPase inactive Spa47 mutants are not able to properly insert the translocon into the host membrane. This seems to be a direct result of an inability of the mutant strains to secrete IpaB and IpaC and properly deliver them to the host cell membrane. Phenotype, overview | Shigella flexneri |
| metabolism | Shigella rely entirely on the actions of a complex T3SS to inject effector proteins into the cytoplasm of host cells, initiating cellular invasion of the colonic epithelium and onset of infection. The Shigella T3SS consists of about 54 genes that reside on a 220-kb virulence plasmid. The entry region of the virulence plasmid contains the mxi, spa, and ipa operons that code for the type III secretion apparatus (T3SA) itself. T3SS ATPases have been identified in several T3SSs | Shigella flexneri |
| additional information | in oligomeric Spa47, ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex, structure analysis and modelling of an activated oligomeric Spa47, overview. ATPase inactive full-length Spa47 point mutants show, that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. Regulation of Spa47 oligomerization in vivo. Key catalytic residues are spatially conserved in Spa47. Generation of an activated hexameric Spa47 model. The predicted active site residues are Lys165 and Glu188, as well as Arg350 | Shigella flexneri |
| physiological function | Shigella virulence is entirely reliant on ATPase active Spa47 and its activity is driven by oligomerization. Type III secretion system ATPase activation and Shigella virulence regulation mechanism by enzyme Spa47, mutational analysis, overview. ATPase inactive full-length Spa47 point mutants show, that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence, structure-function analysis. Active Spa47 is required for proper T3SS translocon formation and Shigella flexneri host cell invasion. ATPase activity is an essential factor in Shigella virulence. Shigella T3SS translocator secretion requires T3SS ATPase activity | Shigella flexneri |
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