HdeA

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0030288 outer membrane-bounded periplasmic space	IEA GO_REF:0000002	ACCEPT	Summary: IEA annotation based on InterPro domain matches (IPR024972, IPR036831). HdeA is well-established as a periplasmic protein with a cleavable signal peptide (residues 1-21) (PMID:8455549, PMID:9298646). This IEA is consistent with and subsumed by the IDA annotation to the same term from PMID:9298646. Reason: Correct localization. HdeA is a secreted periplasmic protein. Multiple studies confirm periplasmic localization including direct protein sequencing from periplasmic fractions (PMID:9298646) and UniProt annotation with signal peptide (residues 1-21). The IEA is redundant with the IDA but not incorrect. Supporting Evidence: PMID:9298646 enriched for proteins based on subcellular location and found several proteins in unexpected subcellular locations
GO:0042597 periplasmic space	IEA GO_REF:0000120	ACCEPT	Summary: IEA annotation from UniProt subcellular location mapping (UniProtKB-SubCell:SL-0200). GO:0042597 "periplasmic space" is a more general term than GO:0030288 "outer membrane-bounded periplasmic space". HdeA is localized to the periplasm as confirmed by multiple experimental studies. Reason: Correct but more general than GO:0030288. The periplasm annotation is well supported by UniProt annotation and experimental evidence. Although the more specific GO:0030288 is also annotated, this broader IEA is not wrong. UniProt function comment states "Periplasm" with evidence from HAMAP-Rule:MF_00946 and PMID:17085547. Supporting Evidence: PMID:17085547 We extracted HdeB from bacteria by the osmotic-shock procedure ...[confirming periplasmic localization of hdeAB operon products]... both proteins are required for optimal protection of the bacterial periplasm against acid stress
GO:0071468 cellular response to acidic pH	IEA GO_REF:0000002	ACCEPT	Summary: IEA annotation from InterPro domain matches. HdeA is a core component of the E. coli acid stress response, activated exclusively at pH below 3 (PMID:15911614). This is a parent term of GO:1990451 "cellular stress response to acidic pH" which is also annotated with experimental evidence. The IEA to this broader term is acceptable as consistent with the more specific experimental annotation. Reason: Correct and well-supported. HdeA is activated by acidic pH and functions specifically in the acid stress response. GO:0071468 is broader than GO:1990451 which is annotated with IMP evidence from PMID:10623550. The broader IEA is not wrong. Supporting Evidence: PMID:15911614 HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3)
GO:1990451 cellular stress response to acidic pH	IEA GO_REF:0000104	ACCEPT	Summary: IEA annotation transferred from manual annotations via shared sequence features (UniRule:UR000106130). GO:1990451 is a child of GO:0071468 "cellular response to acidic pH" and specifically captures the stress response aspect. HdeA is a key effector of the acid stress response, as demonstrated by genetic studies showing hdeA deletion mutants are sensitive to acid stress (PMID:10623550). Reason: Correct annotation. This IEA is consistent with the IMP annotation to the same term from PMID:10623550. HdeA is activated specifically under acid stress conditions (pH < 3) and is required for optimal acid stress protection. Supporting Evidence: PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria
GO:0042802 identical protein binding	IPI PMID:20080625 Protein refolding by pH-triggered chaperone binding and rele...	MARK AS OVER ANNOTATED	Summary: IPI annotation from IntAct based on physical interaction data (HdeA self-interaction). HdeA forms a homodimer at neutral pH that dissociates into active monomers at acidic pH (PMID:10623550, PMID:20080625). The homodimerization is functionally important as the dimer-to-monomer transition is the activation mechanism. However, "identical protein binding" is an uninformative term. The more specific GO:0042803 "protein homodimerization activity" is already annotated with IDA evidence. Reason: GO:0042802 "identical protein binding" is too vague and does not convey meaningful information about HdeA function. The more specific and informative GO:0042803 "protein homodimerization activity" is already annotated (IDA, PMID:10623550). Per curation guidelines, vague binding terms like "protein binding" and "identical protein binding" should be avoided in favor of more informative MF terms. Supporting Evidence: PMID:10623550 HDEA is activated by a dimer-to-monomer transition at acidic pH
GO:0006457 protein folding	IDA PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in...	MODIFY	Summary: IDA annotation for involvement in protein folding from EcoCyc, based on the demonstration that HdeA suppresses aggregation of acid-denatured proteins (PMID:10623550). However, HdeA is primarily a holdase that prevents aggregation rather than actively assisting protein folding. PMID:20080625 later showed that HdeA does facilitate refolding of acid-denatured proteins upon pH neutralization via slow substrate release, but this is a passive mechanism distinct from active foldase activity. Reason: HdeA does not actively catalyze protein folding in the conventional sense (it is ATP- independent and lacks foldase activity). Its primary function is preventing aggregation of acid-denatured proteins (holdase activity). While PMID:20080625 showed it facilitates refolding upon pH neutralization, this is achieved through passive slow release of substrates rather than active folding assistance. The BP term "protein folding" overstates HdeA's role. A more appropriate term would capture the chaperone-mediated protein refolding or protein stabilization aspect. However, given that refolding does occur as a consequence of HdeA activity (PMID:20080625), the annotation is not entirely wrong -- it is the process outcome rather than the mechanism. Proposed replacements: protein refolding Supporting Evidence: PMID:20080625 HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding. PMID:10623550 Functional studies demonstrate that HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins
GO:0044183 protein folding chaperone	EXP PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in...	ACCEPT	Summary: EXP annotation from DisProt for protein folding chaperone activity based on PMID:10623550. The crystal structure study demonstrated that HdeA suppresses aggregation of acid-denatured proteins and suggested chaperone-like functions. GO:0044183 "protein folding chaperone" is defined as "binding to a protein or a protein-containing complex to assist the protein folding process." While HdeA does assist in the overall folding process (preventing aggregation and facilitating refolding upon pH return), it is mechanistically a holdase rather than a foldase. However, GO:0044183 is the best available MF term for chaperone function pending creation of a holdase-specific term. Reason: GO:0044183 is the best available MF term for HdeA's chaperone activity. HdeA binds denatured proteins and assists in the folding process by preventing aggregation and facilitating refolding upon pH neutralization. Although HdeA is mechanistically a holdase (ATP-independent, prevents aggregation in situ), the definition of GO:0044183 ("binding to a protein...to assist the protein folding process") is broad enough to encompass holdase activity. This annotation should be retained as the primary MF annotation pending creation of a holdase-specific GO term. Supporting Evidence: PMID:10623550 We suggest that HDEA may support chaperone-like functions during the extremely acidic conditions PMID:20080625 This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding
GO:0044183 protein folding chaperone	IDA PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in...	ACCEPT	Summary: IDA annotation from DisProt for the same term and reference as the EXP annotation above. This is a duplicate with a different evidence code (IDA vs EXP) from the same source (DisProt) and same reference (PMID:10623550). Both are acceptable as duplicates with different evidence codes are permitted. Reason: Same rationale as the EXP annotation above. GO:0044183 is the best available MF term for HdeA's chaperone function. The IDA evidence code is appropriate given the direct aggregation suppression assays reported in PMID:10623550. Supporting Evidence: PMID:10623550 HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins
GO:0044183 protein folding chaperone	EXP PMID:30573682 Structural basis and mechanism of the unfolding-induced acti...	ACCEPT	Summary: EXP annotation from DisProt based on PMID:30573682. This study used advanced NMR methods to characterize HdeA's activated-state conformation under acidic conditions and identified client-binding sites. It provided structural evidence for the chaperone mechanism: two hydrophobic patches are exposed upon acid-induced unfolding and are essential for client interactions. Reason: PMID:30573682 provides direct structural evidence for HdeA's chaperone function at the atomic level, identifying the client-binding sites and the multistep activation mechanism. GO:0044183 remains the best available MF term. Supporting Evidence: PMID:30573682 the structure of activated HdeA becomes largely disordered and exposes two hydrophobic patches essential for client interactions
GO:0030288 outer membrane-bounded periplasmic space	IDA PMID:9298646 Comparing the predicted and observed properties of proteins ...	ACCEPT	Summary: IDA annotation from EcoCyc based on the Link et al. (1997) proteomics study which identified HdeA by 2-DE and Edman sequencing from periplasmic fractions. The study confirmed that HdeA (then "10K-S") is a periplasmic protein with a cleaved signal peptide. Reason: Well-supported localization. The study used subcellular fractionation and protein identification by sequencing to confirm HdeA is in the periplasm. Additionally confirmed by UniProt signal peptide annotation (residues 1-21) and subsequent studies (PMID:17085547). Supporting Evidence: PMID:9298646 We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone PMID:9298646 We enriched for proteins based on subcellular location
GO:0042803 protein homodimerization activity	IDA PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in...	ACCEPT	Summary: IDA annotation from EcoCyc. The crystal structure of HdeA at 2.0 A resolution (PMID:10623550) revealed that HdeA forms a homodimer at neutral pH. The dimer-to- monomer transition at acidic pH is the activation mechanism for chaperone function. The proteomics study (PMID:9298646) also noted HdeA exists as a "covalent homomultimer." The homodimerization is functionally significant as it represents the inactive storage form. Reason: Accurate and functionally important annotation. HdeA homodimerization is well characterized structurally (PMID:10623550, PMID:9731767) and is directly relevant to the activation mechanism (dimer-to-monomer transition at low pH). This is more informative than the broader GO:0042802 "identical protein binding." Supporting Evidence: PMID:10623550 HDEA is activated by a dimer-to-monomer transition at acidic pH PMID:9298646 Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers
GO:0051082 unfolded protein binding	IDA PMID:15911614 Periplasmic protein HdeA exhibits chaperone-like activity ex...	MODIFY	Summary: IDA annotation from EcoCyc based on PMID:15911614 which demonstrated that HdeA binds acid-denatured proteins at low pH. The study showed HdeA transforms into a disordered conformation at pH below 3 and exposes hydrophobic surfaces that bind denatured substrates, suppressing their aggregation. GO:0051082 "unfolded protein binding" is proposed for obsoletion (go-ontology#30962). HdeA is an ATP-independent, in-situ holdase that prevents aggregation of acid-denatured periplasmic proteins. The most mechanistically appropriate replacement is GO:0140309 "unfolded protein carrier activity," which was created for holdase-type chaperones. However, there is a caveat: GO:0140309 was created specifically for TIM carrier-holdases that escort unfolded proteins between cellular compartments (go-ontology#30552), and its definition requires escort "between two different cellular components." HdeA functions in situ in the periplasm and does not escort proteins between compartments. A general "holdase chaperone activity" NTR would be the ideal replacement (see UNFOLDED_PROTEIN_BINDING.md). Reason: GO:0051082 is proposed for obsoletion. HdeA is a well-characterized holdase: it binds acid-denatured proteins at low pH, prevents their aggregation in the periplasm, and facilitates refolding upon pH neutralization by slow substrate release (PMID:15911614, PMID:20080625). It is ATP-independent, consistent with the periplasm lacking ATP. GO:0140309 "unfolded protein carrier activity" captures the holdase mechanism but its definition strictly requires escort between cellular components, which HdeA does not perform. Until a general holdase NTR is created, GO:0140309 is the closest available term. The existing GO:0044183 annotations also partially capture HdeA's function but from the foldase perspective. Proposed replacements: unfolded protein binding (retain until holdase NTR is created) Supporting Evidence: PMID:15911614 HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3) PMID:15911614 our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values PMID:20080625 HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA
GO:1990451 cellular stress response to acidic pH	IMP PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in...	ACCEPT	Summary: IMP annotation from EcoCyc. PMID:10623550 demonstrated that HdeA supports acid resistance in pathogenic enteric bacteria. The crystal structure study combined functional analysis showing that HdeA is activated at acidic pH and suppresses aggregation of acid-denatured proteins. This is the core biological process for HdeA. Reason: Core biological process annotation. HdeA is a central effector of the cellular stress response to acidic pH. The IMP evidence is appropriate as the study demonstrated the acid-resistance phenotype supported by HdeA. GO:1990451 is a child of GO:0071468 "cellular response to acidic pH" and specifically captures the stress response aspect, which is the relevant context for HdeA function. Supporting Evidence: PMID:10623550 HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria PMID:10623550 HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins
GO:0030288 outer membrane-bounded periplasmic space	RCA PMID:8455549 Function of the Escherichia coli nucleoid protein, H-NS: mol...	ACCEPT	Summary: RCA annotation from EcoCyc based on PMID:8455549 (Yoshida et al., 1993), which originally identified the hdeA gene (then called 10K-S or yhiB) as part of an operon whose expression is enhanced in an hns deletion mutant. The study cloned and sequenced the gene but did not directly demonstrate periplasmic localization experimentally. However, the signal peptide is evident from the sequence. This is consistent with but weaker than the IDA annotation from PMID:9298646. Reason: The RCA evidence is appropriate for sequence-based prediction of periplasmic localization from the identified signal peptide. This annotation is redundant with the IDA from PMID:9298646 but not incorrect. The original identification paper correctly predicted periplasmic localization. Supporting Evidence: PMID:8455549 The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined

Core Functions

Acid-activated periplasmic holdase chaperone that prevents irreversible aggregation of acid-denatured periplasmic proteins during gastric transit (pH < 3)

Molecular Function:

protein folding chaperone

Directly Involved In:

cellular stress response to acidic pH

Cellular Locations:

outer membrane-bounded periplasmic space

Facilitates refolding of acid-denatured periplasmic proteins upon return to neutral pH via slow ATP-independent substrate release

Molecular Function:

protein folding chaperone

Directly Involved In:

protein refolding

Cellular Locations:

outer membrane-bounded periplasmic space

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000104

Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:8455549

Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant.

Original identification of the hdeA gene (10K-S) as part of an operon at 77.5 min whose expression is enhanced in hns deletion mutants.

"The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined"

PMID:9298646

Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12.

Identified HdeA as a highly abundant periplasmic protein by 2-DE and Edman sequencing. Confirmed signal peptide cleavage and periplasmic localization. Noted HdeA exists as a covalent homomultimer.

"We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone ...Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers"

PMID:9731767

Crystal structure of Escherichia coli HdeA.

First crystal structure of HdeA at 2.2 A resolution. Identified the intramolecular disulfide bond (Cys39-Cys87).

PMID:10623550

HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria.

Crystal structure at 2.0 A resolution. Demonstrated HdeA is a homodimer that dissociates at acidic pH. Showed HdeA suppresses aggregation of acid-denatured proteins and supports acid resistance phenotype.

"HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins. We suggest that HDEA may support chaperone-like functions during the extremely acidic conditions"

PMID:15911614

Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation.

Key mechanistic study. HdeA transforms from ordered conformation (inactive, neutral pH) to globally disordered conformation (active, pH < 3). Exposes hydrophobic surfaces for binding denatured substrates. Chaperone activity exclusively within stomach pH range.

"HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3)"

PMID:17085547

Escherichia coli HdeB is an acid stress chaperone.

Demonstrated HdeA and HdeB both required for optimal acid stress protection. HdeA more efficient at pH 2, HdeB at pH 3. Confirmed periplasmic localization.

"both proteins are required for optimal protection of the bacterial periplasm against acid stress...At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA...At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation"

PMID:18359765

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

HdeA promotes solubilization of protein aggregates at neutral pH after acid treatment.

"HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH"

PMID:20080625

Protein refolding by pH-triggered chaperone binding and release.

Demonstrated HdeA facilitates refolding of acid-denatured proteins via pH-triggered binding and release cycle. ATP-independent mechanism: stable binding at low pH prevents aggregation, slow release upon pH neutralization keeps folding intermediates below aggregation threshold.

"HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding"

PMID:21892184

A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance.

Demonstrated cooperation between HdeA and other periplasmic chaperones (DegP, SurA) during acid stress recovery using in vivo crosslinking.

"the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria"

PMID:30573682

Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone.

NMR characterization of activated HdeA. Identified two hydrophobic patches essential for client interactions and three acid-sensitive structural locks regulating activation. Revealed multistep activation mechanism.

"the structure of activated HdeA becomes largely disordered and exposes two hydrophobic patches essential for client interactions...we identified three acid-sensitive regions that act as structural locks in regulating the exposure of the two client-binding sites during the activation process, revealing a multistep activation mechanism"

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