SctN (Spa47, SpaL, MxiB) is the ATPase of the Shigella flexneri type III secretion system (injectisome), encoded in the spa operon of the large virulence plasmid. It is a soluble cytoplasmic enzyme that assembles into a homohexamer forming the central hub of the cytoplasmic sorting platform at the base of the injectisome, where MxiN/SctL forms the radial spokes, Spa33/SctQ the pods and MxiK/SctK links the platform to the basal body. The hexamer sits directly beneath and is aligned with the central channel of the MxiA/SctV export gate. Purified Spa47 is a Mg2+-dependent ATP hydrolase whose activity is switched on by oligomerization: catalytic sites are shared between adjacent protomers, so monomers are only weakly active and the oligomer is roughly eight-fold more active. Catalysis requires a Walker A lysine (Lys165) together with Glu188 and Arg350 and a set of interfacial residues; MxiN/SctL tunes activity in opposite directions on the monomer and the oligomer. ATP hydrolysis by Spa47 is used to recognize secretion substrates, strip their chaperones, unfold them and feed them to the export gate, and is required for building a complete secretion apparatus, for secreting the needle subunits MxiH/MxiI and the translocators IpaB/IpaC, and for epithelial cell invasion. Localization to the injectisome is a property of the N-terminal domain rather than of catalysis, so catalytically dead variants still dock and act as dominant negatives. Spa47 is an evolutionary paralog of the F1-ATP synthase beta subunit and retains the F1 alpha/beta N-terminal domain fold, but it neither synthesizes ATP nor conducts protons; in the injectisome the proton motive force is used by the membrane export gate MxiA/SctV. Its EC number, 7.4.2.8, is the protein-translocating export ATPase reaction.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: Spa47 is a Walker-type P-loop ATPase. Crystal structures of the Shigella protein have been solved with the ATP analog ATPgammaS, a catalytic magnesium ion and an ordered water in the active site, and mutation of the Walker A lysine (Lys165) abolishes activity. Reason: Directly demonstrated for this protein by nucleotide-bound crystal structures and Walker A mutagenesis. ATP hydrolysis activity and protein-exporting ATPase activity are the more informative molecular functions and are also annotated. Supporting Evidence: PMID:30013545 a catalytic magnesium ion and an ordered water molecule PMID:26947936 An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47 |
| GO:0005737 cytoplasm | EXP PMID:18657109 Characterization of soluble complexes of the Shigella flexne... | ACCEPT | Summary: Native Spa47 was characterized directly from the Shigella flexneri cytoplasm, where it is found in two distinct high-molecular-weight soluble complexes with Spa33/SctQ, MxiN/SctL and MxiK/SctK. Reason: Experimental localization of the Shigella protein itself. Spa47 has no transmembrane segment and its injectisome-associated pool sits on the cytoplasmic face of the basal body. Supporting Evidence: PMID:18657109 the native T3SS ATPase, Spa47, from the cytoplasm of Shigella flexneri PMID:18657109 demonstrating it to be in two distinct high-molecular-weight complexes with Spa33: MxiN and MxiK |
| GO:0005737 cytoplasm | EXP PMID:25583506 Visualization of the type III secretion sorting platform of ... | ACCEPT | Summary: Cryo-electron tomography of intact Shigella flexneri injectisomes places the Spa47 hexamer in the cytoplasmic sorting platform at the base of the machine, as the central hub aligned with the MxiA/SctV export gate. Reason: In situ structural evidence for the Shigella protein. It is the more informative of the two cytoplasm rows because it also locates Spa47 within the injectisome-associated sorting platform, but GO has no cellular component term for the T3SS sorting platform; GO:0030257 is the closest available and is separately annotated. Supporting Evidence: PMID:25583506 the hub consists mainly of a hexamer of the Spa47 ATPase PMID:25583506 Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic prediction of the same cytoplasmic location, from the T3SS ATPase domain IPR005714 and the UniProt subcellular location keyword. Reason: Correct, and redundant with the two EXP rows above, which are the primary support. Unlike the other pipeline rows on this entry, this one derives from the T3SS-specific InterPro entry rather than from the shared F1 alpha/beta domain, and it gets the biology right. Supporting Evidence: PMID:18657109 the native T3SS ATPase, Spa47, from the cytoplasm of Shigella flexneri |
| GO:0008564 protein-exporting ATPase activity | IEA GO_REF:0000003 | ACCEPT | Summary: This is the core molecular function of Spa47 and the one the UniProt record itself asserts, via EC 7.4.2.8 (ATP + H2O + protein(side 1) = ADP + phosphate + protein(side 2)). The GO definition covers ATP-driven transfer of a protein across a membrane with concomitant secretion, which is what the T3SS ATPase energizes. Purified Spa47 is a genuine ATPase, and its hydrolytic activity is required for apparatus assembly and for secretion of translocators and effectors. Reason: The most specific and informative molecular function available for this protein, and correctly sourced: the EC mapped here is the export ATPase EC 7.4.2.8, not the ATP synthase EC 7.1.2.2 that mislabels several flagellar FliI orthologs. Supporting Evidence: PMID:26947936 providing the first direct evidence that Spa47 is a bona fide ATPase PMID:31162724 Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47 PMID:27770024 Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence |
| GO:0015986 proton motive force-driven ATP synthesis | IEA GO_REF:0000108 | REMOVE | Summary: This is a GOC logical inference over the TreeGrafter GO:0046933 ATP synthase row, so it stands or falls with that row. Spa47 hydrolyzes ATP; no ATP synthesis by any T3SS ATPase has ever been reported, and the protein has no Fo-type partner or proton channel. The Shigella T3SS does use the proton motive force, but it uses it to translocate substrate through the MxiA/SctV export gate, not to make ATP. Reason: The source molecular function annotation is wrong, so the inferred process is wrong too. Spa47 consumes ATP rather than producing it, and UniProt's own catalytic activity for this entry is the hydrolytic export reaction EC 7.4.2.8. Supporting Evidence: PMID:26947936 providing the first direct evidence that Spa47 is a bona fide ATPase PMID:27171191 MxiA family proteins and their regulators are implicated in utilization of the PMF for protein export PMID:30107569 Substrate translocation itself is mainly fueled by the PMF across the bacterial inner membrane |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: ATP hydrolysis is the catalytic reaction of Spa47, measured directly on the purified Shigella enzyme. Activity is oligomerization-dependent, with the oligomer about eight-fold more active than the monomer because catalytic sites are completed across protomer interfaces. Reason: Correct and experimentally established for this protein. It is retained alongside the more specific GO:0008564 protein-exporting ATPase activity, which states what the hydrolysis is coupled to. Note that this row comes from the T3SS-specific InterPro entry IPR005714, not from the shared F1 domain that produces the erroneous rows on this entry. Supporting Evidence: PMID:26947936 identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer PMID:27770024 each of the tested residues are essential for Spa47 ATPase activity |
| GO:0030254 protein secretion by the type III secretion system | IEA GO_REF:0000002 | ACCEPT | Summary: Spa47 is required for type III secretion in Shigella. A spa47 mutant fails to secrete the needle components MxiH/MxiI and the translocators IpaB/IpaC, and both oligomerization and ATP hydrolysis are needed for a proper secretion profile. Catalytically inactive Spa47 acts as a dominant negative on secretion. Reason: Correct and directly supported by Shigella genetics and phenotyping, well beyond the domain-based inference that generated the row. This is the core biological process of the protein. Supporting Evidence: PMID:27770024 Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence PMID:31162724 Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47 PMID:12864857 we identified interactions between MxiK and Spa33 and Spa47 and between MxiN and Spa33 and Spa47 |
| GO:0030257 type III protein secretion system complex | IEA GO_REF:0000002 | ACCEPT | Summary: Cryo-electron tomography of intact Shigella injectisomes shows the Spa47 hexamer as the central hub of the cytoplasmic sorting platform, in contact with MxiN/SctL, Spa33/SctQ and, through them, the basal body, and aligned with the MxiA/SctV export gate channel. The GO definition of this complex explicitly includes the cytoplasmic ATPase. Reason: Correct, and the best cellular component term available. GO has no term for the T3SS cytoplasmic sorting platform specifically, which would be the precise location of the Spa47 hexamer. Supporting Evidence: PMID:25583506 the hub consists mainly of a hexamer of the Spa47 ATPase PMID:18657109 demonstrating it to be in two distinct high-molecular-weight complexes with Spa33: MxiN and MxiK |
| GO:0042802 identical protein binding | IPI PMID:30460850 Shutting Down Shigella Secretion: Characterizing Small Molec... | ACCEPT | Summary: Spa47 self-association is not incidental: homo-oligomerization is the activation mechanism of the enzyme, because catalytic sites are shared between adjacent protomers. This study purified the active oligomeric species and showed that small-molecule inhibitors reduce its activity without dissolving the oligomer, which required independent characterization of the self-associated state. Reason: An experimental self-interaction that carries real mechanistic information for this protein, so it is not the uninformative protein-binding case. It is a subunit property rather than the core function, which is captured by GO:0008564. Supporting Evidence: PMID:30460850 we leveraged the ability to purify an active oligomeric Shigella T3SS ATPase PMID:30460850 the inhibitors do not function through disruption of Spa47 oligomers or by preventing T3SA formation |
| GO:0042802 identical protein binding | IPI PMID:31162724 Interfacial amino acids support Spa47 oligomerization and sh... | ACCEPT | Summary: This study modeled the activated Spa47 homohexamer, identified two distinct protomer-protomer interfaces, and confirmed them by mutagenesis and crystallography: interface mutants lose both oligomerization and ATP hydrolysis, and lose T3SS secretion and invasion in vivo. Reason: Direct structural and mutational evidence for self-association of this protein, with the interfaces mapped residue by residue. Duplicate of the preceding row in term but independent in evidence. Supporting Evidence: PMID:31162724 we developed a model of an activated Spa47 homo-hexamer PMID:31162724 many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro |
| GO:0046034 ATP metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: This InterPro2GO mapping comes from IPR004100, the F1/V1/A1 alpha/beta N-terminal domain, which Spa47 shares with the rotary ATP synthases by descent. The mapping carries rotary ATP synthase meaning across to an export ATPase. Spa47 hydrolyzes ATP only as the energy source for protein export; it plays no part in cellular ATP synthesis or in ATP turnover as a metabolic pathway. Reason: Literally true in that Spa47 consumes ATP, but uninformative and misleading here. GO captures energy-coupled ATP hydrolysis through the molecular function (GO:0016887, GO:0008564), not as participation in ATP metabolism, and the term's presence alongside the ATP synthase rows reinforces the false impression of ATP synthase biology. Supporting Evidence: PMID:26947936 providing the first direct evidence that Spa47 is a bona fide ATPase PMID:31162724 Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47 |
| GO:0046933 proton-transporting ATP synthase activity, rotational mechanism | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter grafted P0A1C1 onto PTHR15184 at node PTN001807734 and propagated the F1-beta ATP synthase molecular function. Tracing the tree (projects/TREEGRAFTER/rotary_atpase/node_placement.tsv) shows where this goes wrong. The GO:0046933 IBD sits at PTN008558586, whose event type is DUPLICATION. Its two children are PTN008558588, which holds the ATP synthase beta subfamilies (SF51, SF74, SF75, SF76, SF80, SF82, SF83, SF85) and contains every experimental seed for the term, and PTN000390097, a eubacterial node holding the type III export ATPase subfamilies SF9, SF62 and SF81. P0A1C1 is classified in PTHR15184:SF9 (SPI-1 type 3 secretion system ATPase) and its graft node PTN001807734 lies inside PTN000390097. So the assertion was placed one node too deep, at the duplication that created the two paralog lineages, and leaks the ATP synthase function from the F1-beta child into the export ATPase sister clade. Reason: Contradicted by the biology of this protein. Spa47 is a soluble cytoplasmic ATP hydrolase with no transmembrane segment, no Fo-type proton channel and no reported synthetic activity; UniProt assigns it EC 7.4.2.8, the protein-translocating export reaction. Where the Shigella T3SS uses the proton motive force, the protons are handled by the inner-membrane export gate MxiA/SctV, not by the ATPase. The right fix in PAINT is to move the IBD from the duplication node PTN008558586 down to the F1-beta child PTN008558588, which keeps every seeded annotation. Supporting Evidence: PMID:26947936 providing the first direct evidence that Spa47 is a bona fide ATPase PMID:27171191 MxiA family proteins and their regulators are implicated in utilization of the PMF for protein export file:SHIFL/sctN/sctN-notes.md the IBD was placed one node too deep, at the duplication that separates F1-beta from the T3SS/flagellar export ATPases |
| GO:1902600 proton transmembrane transport | IEA GO_REF:0000002 | REMOVE | Summary: Another InterPro2GO mapping from IPR004100, the F1/V1/A1 alpha/beta N-terminal domain. Spa47 is a soluble cytoplasmic protein with no transmembrane segment; cryo-ET places its hexamer in the sorting platform below the membrane, not in it. Efficient type III secretion in Shigella requires the cytoplasmic ATPase and the proton motive force as two separate contributions, and the conserved charged residues implicated in using the proton motive force are in the transmembrane domain of the export gate MxiA/SctV. Reason: The domain-based mapping transfers rotary ATP synthase proton chemistry to an export ATPase that has none. Depending on a proton motive force generated and used elsewhere in the machine is not the same as transporting protons, and no protons cross a membrane through Spa47. Supporting Evidence: PMID:27171191 Efficient T3S requires a cytoplasmic ATPase and the proton motive force (PMF) PMID:30107569 Several conserved charged residues reside within the predicted TMD of SctV that seem to be implicated in utilization of the PMF PMID:25583506 the hub consists mainly of a hexamer of the Spa47 ATPase file:SHIFL/sctN/sctN-deep-research-falcon.md hydrolysis supports substrate recognition, chaperone release and substrate unfolding/presentation to the export gate rather than acting as a membrane transporter itself |
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Download this section (compressed HTML)Q: In PANTHER PTHR15184, the GO:0046933 IBD is asserted at PTN008558586, whose event type is DUPLICATION and whose children are the ATP synthase beta clade (PTN008558588, holding all the experimental seeds) and the eubacterial type III export ATPase clade (PTN000390097, subfamilies SF9, SF62, SF81). P0A1C1 is in SF9 and was grafted at PTN001807734 inside that second clade. Should the IBD be moved down to PTN008558588 so that ATP synthase activity and proton-transporting ATP synthase complex stop propagating into the flagellar and injectisome export ATPases, and should an IRD or NOT be placed at the root of PTN000390097 as well?
Q: The InterPro2GO mapping for IPR004100 (F1/V1/A1 alpha/beta N-terminal domain) contributes GO:0046034 and GO:1902600 to every T3SS and flagellar export ATPase that carries the domain. Since that domain is shared by descent with the rotary ATPases but the export ATPases neither synthesize ATP nor conduct protons, should the IPR004100 mapping be narrowed, or the mapping moved to the rotary-specific InterPro entries?
Q: Would GO benefit from a cellular component term for the type III secretion cytoplasmic sorting platform (the SctN hub, SctL spokes, SctQ pods and SctK linker resolved by cryo-electron tomography)? At present GO:0030257 is the only available term, and it cannot distinguish the sorting platform from the membrane-embedded basal body and export apparatus.
Q: Spa47 hexamerization is required for activity and occurs both free in the cytoplasm and at the injectisome. Should a protein-complex term or a homo-oligomerization process term be used in addition to GO:0042802 identical protein binding to capture the functional hexamer?
Experiment: Test directly whether Spa47 can run in reverse: assay purified oligomeric Spa47 for ATP synthesis from ADP and phosphate, alone and reconstituted into proteoliposomes with an imposed proton gradient, and test the ATPase for sensitivity to F-, V- and P-type ATPase inhibitors as was done for Salmonella FliI. A negative result on this protein would give a citable, organism-specific refutation of the ATP synthase and proton transport propagations rather than relying on the flagellar ortholog.
Type: enzymology and reconstitution
Experiment: Measure Shigella type III secretion rates in strains carrying ATPase-dead Spa47 while independently collapsing the membrane potential and the pH gradient with ionophores, to quantify how much of the export work is done by Spa47 hydrolysis versus the MxiA/SctV proton motive force in this injectisome.
Type: bacterial physiology and secretion assay
Experiment: Determine a cryo-EM structure of the Spa47 hexamer in complex with MxiN/SctL and Spa13/SctO to test whether the SctO stalk occupies the central channel in a gamma-subunit-like manner, and whether there is any rotational coupling, which would settle how far the F1 architectural analogy extends without implying ATP synthase chemistry.
Type: structural biology
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