Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0003755 peptidyl-prolyl cis-trans isomerase activity	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for PPIase activity. SlyD is an FKBP-type PPIase with demonstrated catalytic activity for the cis-trans isomerization of Xaa-Pro bonds (PMID:9188461, PMID:16388577). The PPIase activity is carried in the N-terminal FKBP domain and is conserved across SlyD orthologs. Reason: PPIase activity is one of SlyD's two primary enzymatic activities and is well-supported by both phylogenetic inference and direct experimental evidence (PMID:9188461, PMID:16388577). Supporting Evidence: PMID:9188461 For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment were obtained.
GO:0005829 cytosol	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for cytosol localization. Consistent with IDA evidence from proteomics studies (PMID:15911532, PMID:18304323) and UniProt subcellular location annotation. Reason: Cytosol localization is well-established for SlyD by both phylogenetic inference and proteomics-based experimental evidence.
GO:0042026 protein refolding	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for protein refolding. SlyD has both PPIase and chaperone activities that together promote protein folding and refolding (PMID:16388577, PMID:19356587). The PPIase catalyzes proline-limited refolding steps while the IF domain prevents aggregation of folding intermediates. Reason: SlyD's combined PPIase and chaperone activities make it a bona fide folding helper protein. The protein refolding function is conserved across the SlyD family (PMID:16388577) and supported by IDA evidence. Supporting Evidence: PMID:16388577 All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1).
GO:0003755 peptidyl-prolyl cis-trans isomerase activity	IEA GO_REF:0000120	ACCEPT	Summary: IEA annotation for PPIase activity from combined automated methods. Consistent with the IBA and IDA annotations for the same term. Reason: PPIase activity is a core function supported by multiple evidence types. This broader IEA annotation is acceptable.
GO:0005737 cytoplasm	IEA GO_REF:0000044	ACCEPT	Summary: IEA annotation for cytoplasm localization from UniProt subcellular location mapping. Broader parent of cytosol. UniProt confirms cytoplasmic localization. Reason: Cytoplasmic localization is correct and well-established. The more specific cytosol term is annotated with IDA and IBA evidence.
GO:0016853 isomerase activity	IEA GO_REF:0000043	ACCEPT	Summary: IEA annotation for isomerase activity from UniProt keyword mapping. SlyD is classified as EC 5.2.1.8, which is an isomerase. This is a very broad parent of the more specific PPIase annotation. Reason: As an EC 5.2.1.8 enzyme, SlyD is indeed an isomerase. This broad IEA is acceptable alongside the more specific PPIase annotations.
GO:0042026 protein refolding	IEA GO_REF:0000117	ACCEPT	Summary: IEA annotation for protein refolding from ARBA machine learning models. Consistent with the IBA and IDA annotations for the same term. Reason: Protein refolding is a well-supported function. This IEA annotation is consistent with stronger evidence types.
GO:0046872 metal ion binding	IEA GO_REF:0000043	ACCEPT	Summary: IEA annotation for metal ion binding from UniProt keyword mapping. SlyD binds multiple metal ions (Ni, Zn, Cu, Co) through its C-terminal histidine/cysteine-rich domain (PMID:8300624, PMID:22047179). Reason: Metal ion binding is well-established for SlyD. The more specific nickel, zinc, copper, and cobalt binding annotations are supported by IDA evidence. This broad IEA is acceptable.
GO:0005515 protein binding	IPI PMID:15690043 Interaction network containing conserved and essential prote...	MODIFY	Summary: IPI annotation for protein binding from high-throughput interaction study. The GOA TSV indicates interaction with HypB (P0AAN3) and GrcA (P68066). SlyD-HypB interaction is well-established in the hydrogenase maturation pathway (PMID:15569666, PMID:17426034). Reason: Generic "protein binding" is too vague. The SlyD-HypB interaction is specifically in the context of nickel metallochaperone function for hydrogenase maturation. GO:0170061 "nickel chaperone activity" better captures the functional significance of this interaction. However, for the GrcA interaction, the specific context is less clear. Proposed replacements: nickel chaperone activity
GO:0005515 protein binding	IPI PMID:16412426 Interactions of the Escherichia coli hydrogenase biosyntheti...	MODIFY	Summary: IPI annotation for protein binding from Butland et al. (2006) showing SlyD-HypB complex formation relevant to hydrogenase biosynthesis. Reason: This interaction with HypB is specifically in the context of nickel delivery for [NiFe]-hydrogenase metallocenter assembly. GO:0170061 "nickel chaperone activity" is more informative than generic "protein binding." Proposed replacements: nickel chaperone activity
GO:0005515 protein binding	IPI PMID:19402753 Global functional atlas of Escherichia coli encompassing pre...	MODIFY	Summary: IPI annotation for protein binding from global interaction atlas of E. coli. GOA indicates interaction with HypB (P0AAN3) and GrcA (P68066). Reason: Same reasoning -- the HypB interaction is related to nickel metallochaperone function. GO:0170061 "nickel chaperone activity" is more appropriate. Proposed replacements: nickel chaperone activity
GO:0005515 protein binding	IPI PMID:21185288 The Escherichia coli metal-binding chaperone SlyD interacts ...	MODIFY	Summary: IPI annotation for protein binding from Chung and Zamble (2011) showing SlyD interaction with HypB (P0AAN3) and the hydrogenase 3 large subunit HycE (P16431). SlyD-HycE interaction is mediated by the chaperone domain and is independent of HypB, supporting a direct role in hydrogenase maturation. Reason: These interactions are specifically in the context of [NiFe]-hydrogenase maturation. GO:0170061 "nickel chaperone activity" captures the metallochaperone function. Proposed replacements: nickel chaperone activity Supporting Evidence: PMID:21185288 A SlyD-HycE interaction preceding both iron and nickel insertion to the enzyme was detected, mediated by the chaperone domain of SlyD, and independent of HypB.
GO:0005515 protein binding	IPI PMID:22016389 Protein interactions and localization of the Escherichia col...	MODIFY	Summary: IPI annotation for protein binding from Tao et al. (2012) showing SlyD interaction with HycE (P16431) during nickel insertion to [NiFe] hydrogenase. Reason: Interaction with HycE is in the context of nickel delivery during hydrogenase maturation. GO:0170061 "nickel chaperone activity" is more informative. Proposed replacements: nickel chaperone activity
GO:0000413 protein peptidyl-prolyl isomerization	IDA PMID:9188461 The Escherichia coli SlyD is a metal ion-regulated peptidyl-...	ACCEPT	Summary: IDA annotation for protein peptidyl-prolyl isomerization from Hottenrott et al. (1997). SlyD was demonstrated to catalyze cis/trans isomerization of peptidyl-prolyl bonds with kcat/Km of 29,600 M-1 s-1 for Suc-Ala-Phe-Pro-Arg-4-nitroanilide. The PPIase activity is reversibly inhibited by Ni2+ binding to the C-terminal histidine-rich region. Reason: Direct experimental evidence for the biological process of peptidyl-prolyl isomerization. This is one of SlyD's core catalytic activities. Supporting Evidence: PMID:9188461 the PPIase activity of SlyD is reversibly regulated by binding of three Ni2+ ions to the histidine-rich, C-terminal region
GO:0042026 protein refolding	IDA PMID:16388577 SlyD proteins from different species exhibit high prolyl iso...	ACCEPT	Summary: IDA annotation for protein refolding from Scholz et al. (2006). The study demonstrated that SlyD proteins from multiple species catalyze proline-limited refolding of ribonuclease T1 with very high efficiency and also exhibit pronounced chaperone properties that prevent aggregation. Reason: Strong experimental evidence for SlyD's role in protein refolding. SlyD combines PPIase and chaperone activities that together promote efficient protein folding, making it a bona fide folding helper protein. Supporting Evidence: PMID:16388577 SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity.
GO:0051604 protein maturation	IMP PMID:17426034 The role of complex formation between the Escherichia coli h...	ACCEPT	Summary: IMP annotation for protein maturation from Leach et al. (2007). Mutations in SlyD that disrupt complex formation with HypB result in deficient hydrogenase production in vivo. SlyD stimulates release of nickel from HypB's high-affinity Ni(II)-binding site, and a SlyD truncation lacking the C-terminal metal-binding tail is not functional in vivo for hydrogenase maturation. Reason: Well-supported by mutant phenotype evidence. SlyD is required for proper maturation of the [NiFe]-hydrogenase metallocenter. This is a core in vivo function. Supporting Evidence: PMID:17426034 Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation.
GO:0005515 protein binding	IPI PMID:30758762 Complex formation between the Escherichia coli [NiFe]-hydrog...	MODIFY	Summary: IPI annotation for protein binding from Khorasani-Motlagh et al. (2019) showing SlyD-HypB complex formation with KD of 24 nM. SlyD blocks HypA-HypB interaction. Reason: The SlyD-HypB interaction is specifically in the context of nickel delivery for [NiFe]-hydrogenase maturation. GO:0170061 "nickel chaperone activity" captures the functional significance. Proposed replacements: nickel chaperone activity Supporting Evidence: PMID:30758762 The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP
GO:0005515 protein binding	IPI PMID:32813023 YdiV regulates Escherichia coli ferric uptake by manipulatin...	KEEP AS NON CORE	Summary: IPI annotation for protein binding from Zhang et al. (2020) showing SlyD interaction with Fur (P0A9A9) in the YdiV-SlyD-Fur regulatory pathway for iron homeostasis. SlyD's PPIase activity transforms Fur into a form that does not bind DNA under iron deficiency conditions. Reason: This is a genuine protein-protein interaction, but in a non-core context (iron homeostasis regulation via YdiV-SlyD-Fur pathway). The "protein binding" term is vague, but this interaction represents a secondary regulatory role of SlyD rather than its primary functions. The interaction is biologically meaningful but peripheral to SlyD's core PPIase/chaperone/metallochaperone functions. Supporting Evidence: PMID:32813023 High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner.
GO:0005829 cytosol	IDA PMID:15911532 Localization, annotation, and comparison of the Escherichia ...	ACCEPT	Summary: IDA annotation for cytosol localization from proteomics study by Corbin et al. (2003). Reason: Proteomics-based experimental evidence for cytosol localization.
GO:0005829 cytosol	IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol.	ACCEPT	Summary: IDA annotation for cytosol localization from proteomics study by Ishihama et al. (2008). Reason: Independent proteomics confirmation of cytosol localization.
GO:0005507 copper ion binding	IDA PMID:22047179 Metal selectivity of the Escherichia coli nickel metallochap...	KEEP AS NON CORE	Summary: IDA annotation for copper ion binding from Kaluarachchi et al. (2011). The C-terminal domain of SlyD binds Cu(I) with the highest affinity among all metals tested. However, in vivo, SlyD has a Ni(II)-specific function. Reason: Copper binding is experimentally demonstrated in vitro (PMID:22047179), but the in vivo function of SlyD is specifically as a nickel metallochaperone. The copper binding may not be physiologically relevant, as the same study notes "in vivo studies reveal a Ni(II)-specific function for the protein." Supporting Evidence: PMID:22047179 Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein.
GO:0008270 zinc ion binding	IDA PMID:22047179 Metal selectivity of the Escherichia coli nickel metallochap...	KEEP AS NON CORE	Summary: IDA annotation for zinc ion binding from Kaluarachchi et al. (2011). SlyD binds zinc with affinity similar to nickel in vitro, but in vivo function is nickel-specific. Reason: Zinc binding is experimentally demonstrated (PMID:22047179, PMID:8300624), but SlyD's in vivo metallochaperone function is specific to nickel. Zinc binding may represent a property of the metal-binding domain rather than a specific biological function. Supporting Evidence: PMID:22047179 The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I)
GO:0016151 nickel cation binding	IDA PMID:22047179 Metal selectivity of the Escherichia coli nickel metallochap...	ACCEPT	Summary: IDA annotation for nickel cation binding from Kaluarachchi et al. (2011). SlyD binds multiple Ni(II) ions with submicromolar affinity in the C-terminal domain. Reason: Nickel binding is the physiologically relevant metal-binding function of SlyD, directly related to its role as a nickel metallochaperone for [NiFe]-hydrogenase maturation (PMID:22047179, PMID:19947632). Supporting Evidence: PMID:22047179 in vivo studies reveal a Ni(II)-specific function for the protein.
GO:0050897 cobalt ion binding	IDA PMID:22047179 Metal selectivity of the Escherichia coli nickel metallochap...	KEEP AS NON CORE	Summary: IDA annotation for cobalt ion binding from Kaluarachchi et al. (2011). SlyD binds Co(II) with appreciable affinity but lower than Ni(II) or Zn(II). Reason: Cobalt binding is experimentally demonstrated in vitro but likely not physiologically relevant. In vivo, SlyD functions specifically in nickel homeostasis (PMID:22047179).
GO:0008270 zinc ion binding	IDA PMID:8300624 An Escherichia coli protein consisting of a domain homologou...	KEEP AS NON CORE	Summary: IDA annotation for zinc ion binding from Wuelfing et al. (1994). The original characterization of SlyD showed it binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry. Reason: Zinc binding is experimentally demonstrated but likely not the primary in vivo function. Same reasoning as the other zinc binding annotation. Supporting Evidence: PMID:8300624 The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry, Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly at all.
GO:0016151 nickel cation binding	IDA PMID:19645725 The interaction of the Escherichia coli protein SlyD with ni...	ACCEPT	Summary: IDA annotation for nickel cation binding from Martino et al. (2009). NMR studies showed nickel binding causes conformational rearrangements in the PPIase domain, modulating isomerase activity. Reason: Nickel binding is well-established and directly relevant to regulation of SlyD's PPIase activity and its metallochaperone function. Supporting Evidence: PMID:19645725 the interaction of SlyD with nickel ions entails participation of the novel structural features of the PPIase domain, eliciting structural alterations of the catalytic pocket
GO:0016151 nickel cation binding	IDA PMID:19947632 The Ni(II)-binding properties of the metallochaperone SlyD.	ACCEPT	Summary: IDA annotation for nickel cation binding from Kaluarachchi et al. (2009). ESI-MS showed SlyD binds up to 7 nickel ions in a noncooperative manner with submicromolar affinity. Cysteine residues in the C-terminal domain confer tighter affinity and increased binding capacity. Reason: Detailed characterization of SlyD's nickel binding properties directly related to its metallochaperone function. Supporting Evidence: PMID:19947632 SlyD binds up to seven nickel ions in a noncooperative manner with submicromolar affinity (<2 microM, upper limit)
GO:0016151 nickel cation binding	IDA PMID:8300624 An Escherichia coli protein consisting of a domain homologou...	ACCEPT	Summary: IDA annotation for nickel cation binding from Wuelfing et al. (1994). The original characterization showed tight Ni2+ binding. Reason: The first report of SlyD's nickel binding activity, consistent with subsequent studies showing up to 7 Ni ions bound. Supporting Evidence: PMID:8300624 The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry
GO:0051082 unfolded protein binding	IDA PMID:19356587 NMR solution structure of SlyD from Escherichia coli: spatia...	MODIFY	Summary: IDA annotation for unfolded protein binding from Weininger et al. (2009). NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides. Insulin aggregation is markedly slowed by SlyD. However, this binding is in the context of SlyD's chaperone holdase activity, which prevents aggregation and promotes folding. Reason: SlyD's IF domain functions as a genuine chaperone holdase that binds unfolded proteins, prevents aggregation, and promotes folding (PMID:19356587, PMID:16388577). The term GO:0051082 "unfolded protein binding" describes only the binding aspect. GO:0140309 "unfolded protein carrier activity" better captures the holdase function that prevents aggregation and escorts unfolded substrates. Unlike RidA or CpxP, SlyD's chaperone function is constitutive and a core activity of the IF domain. Proposed replacements: unfolded protein binding (retain until holdase NTR is created) Supporting Evidence: PMID:19356587 NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides. Insulin aggregation is markedly slowed by SlyD* as evidenced by two-dimensional NMR spectroscopy in real time, probably due to SlyD* binding to denatured insulin.
GO:0003755 peptidyl-prolyl cis-trans isomerase activity	IMP PMID:17720786 The peptidyl-prolyl isomerase activity of SlyD is not requir...	ACCEPT	Summary: IMP annotation for PPIase activity from Zhang et al. (2007). Mutations that decrease PPIase activity (I42S, F132Y) do not produce corresponding decreases in hydrogenase production, demonstrating that PPIase activity is separable from the metallochaperone function. Reason: This IMP evidence supports the existence of PPIase activity in SlyD, even though the study's main finding is that PPIase is NOT required for hydrogenase maturation. The mutant phenotype evidence confirms PPIase is a real enzymatic activity of SlyD. Supporting Evidence: PMID:17720786 Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.
GO:0050821 protein stabilization	IMP PMID:17971396 Solubilization of aggregation-prone heterologous proteins by...	KEEP AS NON CORE	Summary: IMP annotation for protein stabilization from Han et al. (2007). SlyD was effective as a fusion partner in preventing aggregation and increasing solubility of aggregation-prone heterologous proteins. SlyD sequesters interactive surfaces of heterologous proteins associated with inclusion body formation. Reason: The protein stabilization function is demonstrated in the context of a biotechnological fusion partner system, which reflects SlyD's chaperone properties. This is a real activity but the in vivo significance as a standalone "protein stabilization" function in E. coli is less clear than its PPIase and metallochaperone roles. Supporting Evidence: PMID:17971396 SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions and the formation of inclusion bodies
GO:0009408 response to heat	IEP PMID:17971396 Solubilization of aggregation-prone heterologous proteins by...	KEEP AS NON CORE	Summary: IEP annotation for response to heat from Han et al. (2007). SlyD synthesis increased 3.37-fold under heat shock conditions compared to non-stress conditions, even when 29 out of 858 soluble proteins were eliminated by the stress. Reason: SlyD upregulation under heat stress is supported by expression profiling, but this is an IEP annotation (expression pattern) rather than functional evidence. The heat stress response may reflect SlyD's general chaperone properties but is not its primary function. Supporting Evidence: PMID:17971396 a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition
GO:0005515 protein binding	IPI PMID:15569666 A role for SlyD in the Escherichia coli hydrogenase biosynth...	MODIFY	Summary: IPI annotation for protein binding from Zhang et al. (2005) showing SlyD-HypB interaction. Sequential peptide affinity purification revealed SlyD in complex with HypB. Deletion of slyD resulted in reduced hydrogenase activity and diminished nickel accumulation. Reason: The SlyD-HypB interaction is specifically in the context of nickel delivery for [NiFe]-hydrogenase metallocenter assembly. GO:0170061 "nickel chaperone activity" is more informative than generic "protein binding." Proposed replacements: nickel chaperone activity Supporting Evidence: PMID:15569666 Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts
GO:0003755 peptidyl-prolyl cis-trans isomerase activity	IDA PMID:9188461 The Escherichia coli SlyD is a metal ion-regulated peptidyl-...	ACCEPT	Summary: IDA annotation for PPIase activity from Hottenrott et al. (1997). Directly measured catalytic activity with kcat/Km of 29,600 M-1 s-1 for Suc-Ala-Phe-Pro-Arg-4-nitroanilide substrate. PPIase activity is of the FKBP type and is reversibly regulated by Ni2+ binding. Reason: Direct enzymatic assay demonstrating PPIase catalytic activity. This is the definitive experimental evidence for SlyD's PPIase function. Supporting Evidence: PMID:9188461 Only when using trypsin instead of chymotrypsin as helper enzyme in the PPIase assay, the enzymatic activity of full-length SlyD and its N-terminal fragment can be measured.
GO:0170061 nickel chaperone activity	IDA PMID:15569666 A role for SlyD in the Escherichia coli hydrogenase biosynth...	NEW	Summary: NEW annotation for nickel chaperone activity. SlyD functions as a nickel metallochaperone that participates in the nickel insertion step of [NiFe]-hydrogenase metallocenter assembly. Deletion of slyD results in reduced hydrogenase activity, which is rescued by excess nickel but not zinc (PMID:15569666). SlyD interacts with HypB and stimulates nickel release from HypB's high-affinity Ni(II)-binding site (PMID:17426034). SlyD also directly interacts with HycE, the hydrogenase 3 large subunit (PMID:21185288). Reason: Nickel metallochaperone activity is one of SlyD's most well-characterized and biologically important functions. GO:0170061 "nickel chaperone activity" (defined as "Directly binding to and delivering nickel ions to a target protein") precisely describes SlyD's role in delivering nickel to [NiFe]-hydrogenase. This function is supported by multiple studies (PMID:15569666, PMID:17426034, PMID:21185288, PMID:22047179) and requires the chaperone domain and C-terminal metal-binding tail but not the PPIase activity (PMID:17720786). Supporting Evidence: PMID:15569666 These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway PMID:17426034 SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation.

Core Functions

SlyD is an FKBP-type PPIase that catalyzes the cis-trans isomerization of Xaa-Pro peptide bonds with high efficiency (kcat/Km approximately 10^6 M-1 s-1 for protein substrates). The PPIase activity is carried in the N-terminal FKBP domain and is reversibly inhibited by Ni2+ binding to the C-terminal region. Together with the chaperone holdase function of the IF domain, the PPIase accelerates slow steps of protein folding.

Molecular Function:

peptidyl-prolyl cis-trans isomerase activity

Directly Involved In:

protein refolding

Cellular Locations:

cytosol

Supporting Evidence:

PMID:9188461
For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km of 29,600 M-1 s-1 for SlyD
PMID:16388577
All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies

SlyD functions as a nickel metallochaperone in the [NiFe]-hydrogenase maturation pathway. It binds up to 7 Ni(II) ions in its C-terminal histidine/cysteine-rich domain, interacts with HypB to stimulate nickel release, and directly interacts with the hydrogenase large subunit HycE to deliver nickel for metallocenter assembly. This function requires the chaperone domain and C-terminal metal-binding tail but NOT the PPIase activity.

Molecular Function:

nickel chaperone activity

Directly Involved In:

protein maturation

Cellular Locations:

cytosol

Supporting Evidence:

PMID:15569666
These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway
PMID:17426034
SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB

The insert-in-flap (IF) domain of SlyD provides chaperone holdase activity that binds unfolded or partially folded proteins, prevents their aggregation, and promotes correct folding. The chaperone activity is spatially separate from the PPIase active site. The IF domain also mediates SlyD's interaction with hydrogenase substrates and Tat signal sequences.

Molecular Function:

unfolded protein binding

Directly Involved In:

protein refolding

Cellular Locations:

cytosol

Supporting Evidence:

PMID:19356587
NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides
PMID:16388577
SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD

References

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000043

Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

GO_REF:0000117

Electronic Gene Ontology annotations created by ARBA machine learning models

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

PMID:8300624

An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif.

SlyD binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry

"The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry, Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly at all."
SlyD consists of an N-terminal FKBP domain and a C-terminal metal-binding domain

"It consists of two domains, of which the first (146 amino acids) shows some homology to the FK506-binding proteins. The second domain (50 amino acids) is extremely rich in potentially metal-binding amino acids, such as histidine, cysteine, and acidic amino acids."

PMID:9188461

The Escherichia coli SlyD is a metal ion-regulated peptidyl-prolyl cis/trans-isomerase.

SlyD is an FKBP-type PPIase with kcat/Km of 29,600 M-1 s-1

"For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment were obtained."
SlyD PPIase activity is reversibly inhibited by nickel binding

"the PPIase activity of SlyD is reversibly regulated by binding of three Ni2+ ions to the histidine-rich, C-terminal region"

PMID:12100551

The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174.

SlyD stabilizes the phiX174 lysis protein E

PMID:15569666

A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway.

SlyD interacts with HypB in the hydrogenase biosynthetic pathway

"The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway."
Deletion of slyD results in reduced hydrogenase activity rescuable by nickel

"Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures"

PMID:15690043

Interaction network containing conserved and essential protein complexes in Escherichia coli.

PMID:15911532

Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth.

PMID:16388577

SlyD proteins from different species exhibit high prolyl isomerase and chaperone activities.

SlyD catalyzes proline-limited refolding with very high efficiency

"All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1)."
SlyD exhibits pronounced chaperone properties for unfolded proteins

"SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity."

PMID:16412426

Interactions of the Escherichia coli hydrogenase biosynthetic proteins: HybG complex formation.

PMID:17426034

The role of complex formation between the Escherichia coli hydrogenase accessory factors HypB and SlyD.

SlyD stimulates nickel release from HypB for hydrogenase maturation

"SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation."
SlyD C-terminal metal-binding tail is required for in vivo hydrogenase function

"a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo"

PMID:17720786

The peptidyl-prolyl isomerase activity of SlyD is not required for maturation of Escherichia coli hydrogenase.

PPIase activity of SlyD is not required for hydrogenase maturation

"Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo."

PMID:17971396

Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive Escherichia coli protein, SlyD.

SlyD is upregulated 3.37-fold under heat shock stress

"a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition"
SlyD facilitates folding and increases solubility of aggregation-prone proteins

"SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions and the formation of inclusion bodies"

PMID:18304323

Protein abundance profiling of the Escherichia coli cytosol.

PMID:19356587

NMR solution structure of SlyD from Escherichia coli: spatial separation of prolyl isomerase and chaperone function.

SlyD IF domain recognizes and binds unfolded proteins

"NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides."
SlyD prevents insulin aggregation via chaperone holdase function

"Insulin aggregation is markedly slowed by SlyD* as evidenced by two-dimensional NMR spectroscopy in real time, probably due to SlyD* binding to denatured insulin."

PMID:19402753

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins.

PMID:19645725

The interaction of the Escherichia coli protein SlyD with nickel ions illuminates the mechanism of regulation of its peptidyl-prolyl isomerase activity.

Nickel binding causes conformational changes in PPIase domain regulating activity

"the interaction of SlyD with nickel ions entails participation of the novel structural features of the PPIase domain, eliciting structural alterations of the catalytic pocket"

PMID:19947632

The Ni(II)-binding properties of the metallochaperone SlyD.

SlyD binds up to 7 nickel ions noncooperatively with submicromolar affinity

"SlyD binds up to seven nickel ions in a noncooperative manner with submicromolar affinity (<2 microM, upper limit)"
Cysteine residues in C-terminal domain confer tighter Ni affinity

"the characterization of SlyD mutants demonstrates that the cysteine residues in the C-terminal domain confer tighter affinity as well as increased binding capacity to SlyD"

PMID:21185288

The Escherichia coli metal-binding chaperone SlyD interacts with the large subunit of [NiFe]-hydrogenase 3.

SlyD directly interacts with HycE via its chaperone domain

"A SlyD-HycE interaction preceding both iron and nickel insertion to the enzyme was detected, mediated by the chaperone domain of SlyD, and independent of HypB."

PMID:22016389

Protein interactions and localization of the Escherichia coli accessory protein HypA during nickel insertion to [NiFe] hydrogenase.

PMID:22047179

Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD.

SlyD binds Ni, Zn, Cu, Co but has Ni-specific in vivo function

"Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein."
Metal affinity order is Mn,Fe < Co < Ni ~ Zn << Cu

"The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I)"

PMID:30758762

Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel maturation factors.

SlyD-HypB complex has KD of 24 nM

"The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP"
SlyD blocks HypA-HypB interaction

"A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments."

PMID:32813023

YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of Fur in a SlyD-dependent manner.

SlyD participates in iron homeostasis regulation via YdiV-SlyD-Fur pathway

"High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner."

📚 Additional Documentation

Deep Research Bioreason

(SlyD-deep-research-bioreason.md)

provider: bioreason-pro
model: RL
source: app.bioreason.net
date: '2026-03-21'
uniprot_id: P0A9K9
gene: SlyD
organism: Escherichia coli

Thinking Trace

I begin with the InterPro architecture. The sequence is dominated by IPR046357 (Peptidyl-prolyl cis-trans isomerase domain superfamily, residues 1–110), indicating the entire polypeptide adopts a canonical PPIase fold. Nested within this superfamily, IPR001179 (FKBP-type peptidyl-prolyl cis-trans isomerase domain, residues 4–66) defines a compact FKBP catalytic core. The ordered layout—an FKBP catalytic module embedded in a full-length PPIase scaffold—creates a hydrophobic active site pocket that binds proline-containing peptides and lowers the transition-state barrier between cis and trans imidic bonds. This architecture causes peptidyl-prolyl cis-trans isomerase chemistry, establishing the molecular function formalized as GO:0003755 peptidyl-prolyl cis-trans isomerase activity.

Catalysis of prolyl isomerization accelerates rate-limiting folding steps in many substrates. By rapidly equilibrating the prolyl bond, the enzyme relieves kinetic bottlenecks and promotes productive folding pathways. This causally links the FKBP-type catalytic core to the biological process of GO:0006457 protein folding, where assisting assembly and maturation of diverse polypeptides is central.

The absence of signal peptides or transmembrane segments and the soluble FKBP fold argue for a cytosolic residency. Such soluble folding catalysts operate where nascent chains emerge and where cytosolic chaperone systems function; thus the cellular component is the cytoplasm, corresponding to GO:0005737 cytoplasm.

Mechanistically, the FKBP core binds and isomerizes proline-imidic bonds through a transient hydrophobic cavity, cycling between substrate capture and release to isomerize diverse client peptides. In the bacterial cytoplasm, this activity likely cooperates with core proteostasis machinery. I hypothesize transient assemblies with GroEL/GroES to receive partially folded clients, with Trigger Factor at ribosomes to triage nascent chains, and with the DnaK-DnaJ-GrpE system to hand off substrates. Such partnerships would position the enzyme at the hub of cytosolic protein biogenesis and quality control.

Functional Summary

A soluble cytoplasmic folding catalyst in Escherichia coli that accelerates the rate-limiting isomerization of proline bonds in peptide substrates. By rapidly equilibrating cis and trans conformations of proline-containing segments, it expedites cytosolic protein folding and integrates into the proteostasis network that manages maturation and assembly of diverse client proteins.

UniProt Summary

PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic bonds in oligopeptides.

InterPro Domains

Domain	Type	Range
IPR046357	Peptidyl-prolyl cis-trans isomerase domain superfamily	homologous_superfamily [1-110]
IPR001179	FKBP-type peptidyl-prolyl cis-trans isomerase domain	domain [4-66]

GO Term Predictions

Molecular Function

GO:0003755 Peptidyl-prolyl cis-trans isomerase activity
GO:0016859 Cis-trans isomerase activity
GO:0140096 Catalytic activity, acting on a protein
GO:0016853 Isomerase activity
GO:0003824 Catalytic activity

Biological Process

GO:0006457 Protein folding

Cellular Component

GO:0005737 Cytoplasm
GO:0005829 Cytosol
GO:0005622 Intracellular anatomical structure

Deep Research Bioreason Full

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BioReason Chat Export

Exported on March 21, 2026 at 09:27 PM

Organism: Escherichia coli (strain K12)

Sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGCCGGHGHDHGHEHGGEGCCGGKGNGGCGCH

Thinking Trace

I begin with the InterPro architecture. The polypeptide is fully encompassed by IPR046357 (Peptidyl-prolyl cis-trans isomerase domain superfamily, residues 1–162), establishing a canonical PPIase scaffold. Within its N-terminal half sits IPR001179 (FKBP-type peptidyl-prolyl cis-trans isomerase domain, residues 4–95), which supplies the catalytic FKBP fold that accelerates cis-trans isomerization of peptidyl-proline bonds. Embedded in the mid-region is IPR048261 (PPIase chaperone SlpA/SlyD-like, insertion domain superfamily, residues 79–121). This insertion module is characteristic of bacterial PPIase chaperones and creates an auxiliary substrate-binding surface that enhances affinity for unfolded or metastable polypeptides and coordinates metal-assisted folding typical of SlyD/SlpA-like proteins. The ordered layout—an FKBP catalytic core followed by a SlyD-like insertion—causes robust peptidyl-prolyl isomerase chemistry coupled to holdase/chaperone activity.

This FKBP-type core dictates the molecular function: it catalyzes peptidyl-prolyl cis-trans isomerization (GO:0003755), a rate-limiting step in folding that the FKBP architecture accomplishes by transiently stabilizing the twisted amide transition state. The SlyD-like insertion domain broadens substrate scope and stabilizes intermediate conformations, ensuring efficient isomerase-driven maturation.

From this chemistry, the biological role follows. By accelerating proline isomerization and stabilizing intermediates, the protein promotes client attainment of native structure; this places it squarely in protein folding (GO:0006457). The chaperone-enhancing insertion domain further supports a folding pathway that operates independently of Hsp70/Hsp40 when needed, yet functionally interfaces with general proteostasis.

Cellular localization is inferred from the soluble FKBP architecture and absence of transmembrane segments or secretion signals, pointing to a cytosolic residence. In bacteria, soluble folding catalysts concentrate where nascent chains and stress-unfolded substrates accumulate—namely the cytoplasm—which aligns with cytoplasm (GO:0005737).

Mechanistically, the protein likely binds unfolded or partially folded polypeptides via the SlyD-like insertion, then uses the FKBP catalytic center to resolve proline isomerization barriers. It probably transiently forms complexes with other proteostasis factors. In Escherichia coli, plausible partners include the GroEL/GroES chaperonin system that receives folded clients, triggerosome components that triage aggregation-prone species, and the DnaK-DnaJ-GrpE network that buffers misfolded intermediates. Such associations would route clients either toward productive folding or toward degradation when refolding fails, consistent with a cytosolic folding hub.

Functional Summary

A cytosolic peptidyl-prolyl isomerase that accelerates protein folding by catalyzing cis-trans interconversion at proline bonds and by acting as a soluble folding chaperone. Its FKBP catalytic core drives isomerization, while an auxiliary insertion module enhances affinity for unfolded substrates and supports holdase/chaperone activity. Operating in the bacterial cytoplasm, it collaborates with general proteostasis pathways to stabilize folding intermediates and channel clients toward productive maturation or quality-control pathways.

UniProt Summary

PPIases accelerate the folding of proteins (By similarity).

InterPro Domains

Peptidyl-prolyl cis-trans isomerase domain superfamily (IPR046357, homologous_superfamily) — residues 1-162
FKBP-type peptidyl-prolyl cis-trans isomerase domain (IPR001179, domain) — residues 4-95
PPIase chaperone SlpA/SlyD-like, insertion domain superfamily (IPR048261, homologous_superfamily) — residues 79-121

GO Terms

Molecular Function: molecular_function (GO:0003674), binding (GO:0005488), catalytic activity (GO:0003824), isomerase activity (GO:0016853), ion binding (GO:0043167), catalytic activity, acting on a protein (GO:0140096), protein binding (GO:0005515), cis-trans isomerase activity (GO:0016859), unfolded protein binding (GO:0051082), cation binding (GO:0043169), peptidyl-prolyl cis-trans isomerase activity (GO:0003755), metal ion binding (GO:0046872), transition metal ion binding (GO:0046914), zinc ion binding (GO:0008270), copper ion binding (GO:0005507), nickel cation binding (GO:0016151), cobalt ion binding (GO:0050897)

Biological Process: biological_process (GO:0008150), metabolic process (GO:0008152), biological regulation (GO:0065007), response to stimulus (GO:0050896), biological process involved in interspecies interaction between organisms (GO:0044419), cellular process (GO:0009987), response to abiotic stimulus (GO:0009628), regulation of biological quality (GO:0065008), modulation by symbiont of host cellular process (GO:0044068), nitrogen compound metabolic process (GO:0006807), protein folding (GO:0006457), modulation of process of another organism (GO:0035821), response to stress (GO:0006950), organic substance metabolic process (GO:0071704), biological process involved in symbiotic interaction (GO:0044403), primary metabolic process (GO:0044238), response to heat (GO:0009408), modulation by symbiont of host signal transduction pathway (GO:0052027), response to temperature stimulus (GO:0009266), modulation of signal transduction in another organism (GO:0044501), biological process involved in interaction with host (GO:0051701), protein maturation by protein folding (GO:0022417), organonitrogen compound metabolic process (GO:1901564), protein refolding (GO:0042026), protein metabolic process (GO:0019538), modulation by symbiont of host process (GO:0044003), regulation of protein stability (GO:0031647), macromolecule metabolic process (GO:0043170), protein modification process (GO:0036211), macromolecule modification (GO:0043412), protein maturation (GO:0051604), protein stabilization (GO:0050821), gene expression (GO:0010467), peptidyl-amino acid modification (GO:0018193), peptidyl-proline modification (GO:0018208), protein peptidyl-prolyl isomerization (GO:0000413)

Cellular Component: cellular_component (GO:0005575), cellular anatomical entity (GO:0110165), cytoplasm (GO:0005737), cytosol (GO:0005829), intracellular anatomical structure (GO:0005622)

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Deep Research Bioreason Rl

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Exported on March 21, 2026 at 09:27 PM

Organism: Escherichia coli (strain K12)

Sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGCCGGHGHDHGHEHGGEGCCGGKGNGGCGCH

Thinking Trace

Functional Summary

UniProt Summary

PPIases accelerate the folding of proteins (By similarity).

InterPro Domains

Peptidyl-prolyl cis-trans isomerase domain superfamily (IPR046357, homologous_superfamily) — residues 1-162
FKBP-type peptidyl-prolyl cis-trans isomerase domain (IPR001179, domain) — residues 4-95
PPIase chaperone SlpA/SlyD-like, insertion domain superfamily (IPR048261, homologous_superfamily) — residues 79-121

GO Terms

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Deep Research Bioreason Truncated

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Exported on March 21, 2026 at 09:49 PM

Organism: Escherichia coli

Sequence:

MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANMYQNDDLQRHGGYAVDQHGVIALMKVHKEKNNTLTLTEIAEYNKAFFDKINPQDASKVFDDVKLDKYITEGSKQVVLILKNGKLYDDFKKLKPQVEKFYDALKVTLKNIKPTDQKEEHLRTYMSEHKFYNAIAMAKIDDLQYRQKAADTGMDIKDIT

Thinking Trace

Functional Summary

UniProt Summary

PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic bonds in oligopeptides.

InterPro Domains

Peptidyl-prolyl cis-trans isomerase domain superfamily (IPR046357, homologous_superfamily) — residues 1-110
FKBP-type peptidyl-prolyl cis-trans isomerase domain (IPR001179, domain) — residues 4-66

GO Terms

Molecular Function: molecular_function (GO:0003674), catalytic activity (GO:0003824), isomerase activity (GO:0016853), catalytic activity, acting on a protein (GO:0140096), peptidyl-prolyl cis-trans isomerase activity (GO:0003755), cis-trans isomerase activity (GO:0016859)

Biological Process: biological_process (GO:0008150), cellular process (GO:0009987), protein folding (GO:0006457)

Generated by BioReason

Deep Research Falcon

(SlyD-deep-research-falcon.md)

provider: falcon
model: Edison Scientific Literature
cached: false
start_time: '2026-03-22T17:57:47.951915'
end_time: '2026-03-22T18:07:45.073877'
duration_seconds: 597.12
template_file: templates/gene_research_go_focused.md
template_variables:
organism: ECOLI
gene_id: SlyD
gene_symbol: slyD
uniprot_accession: P0A9K9
protein_description: 'RecName: Full=FKBP-type peptidyl-prolyl cis-trans isomerase
SlyD; Short=PPIase; EC=5.2.1.8; AltName: Full=Histidine-rich protein; AltName:
Full=Metallochaperone SlyD; AltName: Full=Rotamase; AltName: Full=Sensitivity
to lysis protein D; AltName: Full=WHP;'
gene_info: Name=slyD; OrderedLocusNames=b3349, JW3311;
organism_full: Escherichia coli (strain K12).
protein_family: Belongs to the FKBP-type PPIase family. .
protein_domains: PPIase_dom_sf. (IPR046357); PPIase_FKBP_dom. (IPR001179); SlpA/SlyD-like_ins_sf.
(IPR048261); FKBP_C (PF00254)
provider_config:
timeout: 600
max_retries: 3
parameters:
allowed_domains: []
temperature: 0.1
citation_count: 31

Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: P0A9K9
Protein Description: RecName: Full=FKBP-type peptidyl-prolyl cis-trans isomerase SlyD; Short=PPIase; EC=5.2.1.8; AltName: Full=Histidine-rich protein; AltName: Full=Metallochaperone SlyD; AltName: Full=Rotamase; AltName: Full=Sensitivity to lysis protein D; AltName: Full=WHP;
Gene Information: Name=slyD; OrderedLocusNames=b3349, JW3311;
Organism (full): Escherichia coli (strain K12).
Protein Family: Belongs to the FKBP-type PPIase family. .
Key Domains: PPIase_dom_sf. (IPR046357); PPIase_FKBP_dom. (IPR001179); SlpA/SlyD-like_ins_sf. (IPR048261); FKBP_C (PF00254)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "slyD" matches the protein description above
Verify the organism is correct: Escherichia coli (strain K12).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'slyD' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information

Research Target:

Please provide a comprehensive research report on the gene slyD (gene ID: SlyD, UniProt: P0A9K9) in ECOLI.

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Output

Question: You are an expert researcher providing comprehensive, well-cited information.

Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies

Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: P0A9K9
Protein Description: RecName: Full=FKBP-type peptidyl-prolyl cis-trans isomerase SlyD; Short=PPIase; EC=5.2.1.8; AltName: Full=Histidine-rich protein; AltName: Full=Metallochaperone SlyD; AltName: Full=Rotamase; AltName: Full=Sensitivity to lysis protein D; AltName: Full=WHP;
Gene Information: Name=slyD; OrderedLocusNames=b3349, JW3311;
Organism (full): Escherichia coli (strain K12).
Protein Family: Belongs to the FKBP-type PPIase family. .
Key Domains: PPIase_dom_sf. (IPR046357); PPIase_FKBP_dom. (IPR001179); SlpA/SlyD-like_ins_sf. (IPR048261); FKBP_C (PF00254)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "slyD" matches the protein description above
Verify the organism is correct: Escherichia coli (strain K12).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Please provide a comprehensive research report on the gene slyD (gene ID: SlyD, UniProt: P0A9K9) in ECOLI.

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Research report: Escherichia coli K-12 slyD (UniProt P0A9K9) — functional annotation

0) Target verification (critical identity check)

The literature summarized here consistently describes SlyD in Escherichia coli as (i) an FKBP-type peptidyl‑prolyl cis/trans isomerase (PPIase; EC 5.2.1.8), (ii) possessing an insert-in-flap (IF) chaperone domain, and (iii) containing a C-terminal histidine/cysteine-rich metal-binding tail that binds Ni(II) and can regulate PPIase activity—matching the UniProt P0A9K9 description and domain architecture (erdmann2007thenickel‐regulatedpeptidyl pages 1-5, zhang2005arolefor pages 1-1, lindemeier2024positivechargespromote pages 1-3).

1) Key concepts and current understanding (definitions and molecular function)

1.1 Peptidyl-prolyl cis/trans isomerase (PPIase) activity

Definition. PPIases catalyze the cis/trans isomerization of peptidyl‑prolyl bonds, a reaction that can be rate-limiting in protein folding. SlyD is an FKBP-family PPIase in E. coli (erdmann2007thenickel‐regulatedpeptidyl pages 1-5, zhang2005arolefor pages 1-1).

Substrate specificity and kinetics. In the classical protease-coupled chromogenic tetrapeptide assay (Suc-Ala-X-Pro-Arg-4-nitroanilide), E. coli SlyD shows measurable catalytic efficiency; for Suc-Ala-Phe-Pro-Arg-4NA, reported kcat/KM ≈ 29,600 M−1 s−1 for full-length SlyD and 18,600 M−1 s−1 for an N‑terminal fragment lacking the histidine-rich tail (erdmann2007thenickel‐regulatedpeptidyl pages 1-5). These data support that the PPIase catalytic core is N‑terminal, while the C-terminal tail is dispensable for basal peptide PPIase activity (erdmann2007thenickel‐regulatedpeptidyl pages 1-5).

1.2 Chaperone / folding-catalyst behavior (IF domain)

Beyond catalysis on short peptides, SlyD functions as a protein-folding catalyst with a chaperone-like binding site that recognizes unfolded states. Protein-substrate assays show much higher apparent efficiency than tetrapeptide assays; one excerpt reports for a protein refolding substrate (RNase T1 refolding) a kcat/KM ≈ 6.8 × 10^6 M−1 s−1 with KM ≈ 1.65 μM, and notes an ~1000-fold higher efficiency on protein versus peptide substrates (burk2007possiblerolesof pages 22-25). In addition, the IF domain can be the dominant determinant for binding and protein-substrate handling (lindemeier2024positivechargespromote pages 3-4, lindemeier2024positivechargespromote pages 14-16).

1.3 Metal binding and metal-dependent regulation

SlyD is notable among FKBPs for being transition-metal regulated. It contains a C-terminal metal-binding region rich in residues capable of coordinating metals; one study explicitly characterizes this region in E. coli as containing ~15 histidines, 6 cysteines, and 7 acidic residues (Asp/Glu) in an ~50-residue segment (zhang2005arolefor pages 1-1).

Nickel regulation of PPIase activity. Ni(II) binding can strongly and reversibly inhibit PPIase activity; one excerpt reports that 1 μM SlyD + 50 μM NiCl2 causes approximately a 90% decrease in PPIase activity (burk2007possiblerolesof pages 22-25). Another primary study reports reversible regulation by binding of three Ni2+ ions to the C-terminal histidine-rich region (erdmann2007thenickel‐regulatedpeptidyl pages 1-5). Structural/biophysical evidence further supports that nickel binding leads to conformational changes around the catalytic pocket (kaluarachchi2012slydani pages 59-62).

Metal-binding constants/stoichiometry (reported but incompletely consolidated across sources). One excerpt summarizes three metal-binding sites with association constants ~9.5 × 10^5, 4.9 × 10^5, and 4.4 × 10^5 M−1, and notes binding of divalent metals including Ni2+, Zn2+, Cu2+, Co2+ (burk2007possiblerolesof pages 22-25). Because this comes from a secondary excerpt rather than the original primary measurement, it should be treated as a synthesized summary rather than a definitive consensus.

2) Biological role(s), pathways, and cellular location

2.1 [NiFe]-hydrogenase maturation and nickel trafficking (cytosolic maturation pathway)

A well-supported in vivo role for SlyD in E. coli is in [NiFe]-hydrogenase biosynthesis, particularly affecting the nickel insertion step.

Protein–protein interaction with HypB. SlyD was identified in a complex with HypB (an accessory protein required for hydrogenase active-site assembly), and the association was confirmed by chemical cross-linking of purified proteins (zhang2005arolefor pages 1-1).

Genetic phenotype. Deleting slyD causes a marked reduction of hydrogenase activity in extracts from anaerobically grown cells; in-gel assays showed diminished activities of hydrogenase 1 and 2, and Western analysis showed decreased processing of HycE (hydrogenase-3 large subunit) consistent with defective active-site cofactor assembly (zhang2005arolefor pages 1-1). The hydrogenase maturation defects were rescued by growth in excess nickel, supporting a nickel-trafficking/insertion role (zhang2005arolefor pages 1-1).

Nickel accumulation phenotype (quantitative). Radioactive 63Ni accumulation assays showed that ΔslyD cells accumulate ~half as much nickel as wild type, and that SlyD overexpression increases nickel accumulation (zhang2005arolefor pages 5-6). The associated figure evidence is shown in Zhang et al. Figure 6 (zhang2005arolefor media 40dc7d88).

Interpretation (expert synthesis in the primary study). Together, these results were interpreted to indicate SlyD contributes to nickel delivery/storage and insertion during hydrogenase maturation (zhang2005arolefor pages 1-1, zhang2005arolefor pages 5-6).

Cellular location. The described interaction partners (HypB; HycE maturation defects) and assays (cytoplasmic extracts) support that the relevant hydrogenase-maturation function of SlyD occurs in the cytosol (zhang2005arolefor pages 1-1).

2.2 Tat signal peptide/substrate recognition (2024 development)

A major recent development (2024) is a mechanistic clarification of how E. coli SlyD recognizes Tat (twin-arginine translocation) signal peptides, suggesting a chaperone role upstream of translocation.

Binding rules (conceptual). Peptide array work found SlyD binds positively charged peptides, with a preference for arginines over lysines, and that adjacent hydrophobic residues enhance binding—especially around the twin-arginine motif characteristic of Tat signals (lindemeier2024positivechargespromote pages 1-3).

Quantitative thermodynamics (ITC). Using ITC with a model Tat substrate signal peptide (HiPIP), the study reports 1:1 binding stoichiometry, and that mutation of the twin arginines to glutamines reduces affinity by ≥100-fold, with an example KD = 38.7 ± 2 μM for the QQ variant (lindemeier2024positivechargespromote pages 9-11). The RR- and KK-variants show enthalpy-driven binding with a stronger preference for RR (lindemeier2024positivechargespromote pages 9-11).

Structural determinants on SlyD (mutagenesis). Mutations in the IF-domain hydrophobic pocket (e.g., F84 and F96) strongly reduce binding; a double mutant F84N/F96N abolished measurable binding in ITC, while mutation of an FKBP catalytic residue (Y68S) did not change KD, supporting the IF domain as the principal recognition module for Tat signal peptides (lindemeier2024positivechargespromote pages 9-11).

Mechanistic model. The authors propose a two-step mechanism in which electrostatic attraction to a negatively charged IF-domain surface precedes induced-fit engagement of a hydrophobic pocket, consistent with many chaperone recognition models (lindemeier2024positivechargespromote pages 14-16).

3) Recent developments and latest research (prioritizing 2023–2024)

2024 (PLOS ONE): charge-driven Tat signal recognition. The 2024 Lindemeier et al. paper provides the clearest recent, quantitative update for E. coli SlyD function: defining binding specificity for Tat signals and mapping the IF-domain residues that determine affinity, using peptide arrays and ITC (lindemeier2024positivechargespromote pages 1-3, lindemeier2024positivechargespromote pages 9-11).

Gap note (2023–2024 for hydrogenase maturation). Within the retrieved corpus, there are not clearly retrieved 2023–2024 primary studies adding new mechanistic detail for E. coli SlyD’s hydrogenase-related nickel trafficking beyond the established 2005 work; therefore, the hydrogenase maturation portion of this report necessarily leans on foundational work while highlighting that the Tat-chaperone role has a more recent (2024) quantitative update (zhang2005arolefor pages 1-1, lindemeier2024positivechargespromote pages 1-3).

4) Current applications and real-world implementations

4.1 SlyD as a solubility/folding fusion partner in recombinant expression

SlyD has been engineered as an N-terminal fusion tag to improve the cytoplasmic solubility of aggregation-prone proteins.

Stress responsiveness and rationale. One study reports SlyD was identified as a stress-responsive, aggregation-resistant protein: SlyD synthesis increased ~3.37-fold under heat shock, while many soluble proteins decreased strongly (han2007solubilizationofaggregationprone pages 1-2).

Implementation details. SlyD fusions were expressed in E. coli BL21(DE3), including constructs cloned into pT7-7, and purified using Ni2+-IMAC when His-tagged; one detailed workflow includes binding buffer composition (50 mM potassium phosphate, 300 mM KCl, 20 mM imidazole, pH 7.0) and processing steps for inclusion bodies and soluble fractions (han2007solubilizationofaggregationprone pages 2-3).

Demonstrated outcomes. SlyD fusion expression increased solubility for multiple aggregation-prone proteins and enabled recovery of correctly folded products; for example, a (His)6-SlyD fusion strategy yielded recombinant G-CSF with a CD spectrum consistent with correct folding (han2007solubilizationofaggregationprone pages 4-6).

4.2 SlyD as a major contaminant in His-tag metal affinity purification (IMAC)

Because SlyD is histidine-rich and metal-binding, it is a known endogenous contaminant that can co-purify in Ni/Co IMAC workflows.

A practical study on recombinant purification explicitly identifies SlyD as a recurrent IMAC contaminant and describes an engineered expression strain (LOBSTR) carrying genomic modifications of slyD (and arnA) to reduce binding to Ni/Co resins, enabling cleaner single-step IMAC—particularly important for low-yield proteins (typical yields noted 0.1–1 mg/L) (andersen2013optimizede.coliexpressionstrain pages 4-5).

4.3 Use of SlyD* fusions in diagnostic immunoassays

SlyD truncations (SlyD) have been used as fusion partners to produce soluble oligomeric antigens (e.g., gp41 fusions) applied in double-antigen sandwich immunoassays (Elecsys 2010), using antigen concentrations such as 750 ng/mL* (scholz2006slydproteinsfrom pages 9-10).

5) Expert opinion and synthesis (authoritative analysis)

5.1 Multi-function protein with separable but coupled modules

The collective evidence supports a model where E. coli SlyD is modular: an FKBP catalytic PPIase domain, an IF chaperone domain that drives client recognition, and a C-terminal metal-binding tail that can tune activity and participate in nickel trafficking (lindemeier2024positivechargespromote pages 1-3, erdmann2007thenickel‐regulatedpeptidyl pages 1-5, zhang2005arolefor pages 1-1).

5.2 Mechanistic plausibility for coupling nickel biology to folding control

The reversible Ni2+-dependent inhibition of PPIase activity suggests a potential mechanism for linking enzyme activity to intracellular nickel availability (erdmann2007thenickel‐regulatedpeptidyl pages 1-5, burk2007possiblerolesof pages 22-25). However, the physiological interpretation remains nuanced: in hydrogenase maturation, deletion phenotypes and nickel rescue imply SlyD mainly acts in nickel delivery/handling rather than needing PPIase catalysis per se (zhang2005arolefor pages 1-1, zhang2005arolefor pages 5-6).

5.3 Emergent chaperone role in Tat pathway substrate handling (2024)

The 2024 ITC/mutagenesis data strongly support that SlyD’s IF domain is a signal peptide recognition module with specificity rules aligned to Tat biology (twin-arginine motif, adjacent hydrophobicity), providing a plausible role as a chaperone that binds Tat signal peptides and possibly prevents off-pathway aggregation or degradation prior to Tat-mediated transport (lindemeier2024positivechargespromote pages 1-3, lindemeier2024positivechargespromote pages 9-11, lindemeier2024positivechargespromote pages 14-16).

6) Key quantitative statistics and data points (selected)

PPIase peptide substrate (Suc-Ala-Phe-Pro-Arg-4NA): kcat/KM 29,600 M−1 s−1 (full-length) and 18,600 M−1 s−1 (N-terminal fragment) (erdmann2007thenickel‐regulatedpeptidyl pages 1-5).
Protein substrate refolding (example reported): kcat/KM 6.8 × 10^6 M−1 s−1, KM 1.65 μM (burk2007possiblerolesof pages 22-25).
Ni2+ inhibition of PPIase: ~90% activity decrease at 1 μM SlyD + 50 μM NiCl2 (burk2007possiblerolesof pages 22-25).
Tat signal peptide binding (ITC): HiPIP signal peptide binds 1:1; RR→QQ reduces affinity ≥100-fold; example KD = 38.7 ± 2 μM for QQ variant (lindemeier2024positivechargespromote pages 9-11).
Nickel accumulation phenotype: ΔslyD accumulates ~half as much 63Ni as wild type; overexpression increases nickel accumulation (zhang2005arolefor pages 5-6, zhang2005arolefor media 40dc7d88).

Evidence summary table

The following table consolidates major functional annotation claims, quantitative data, experimental basis, and primary sources.

Function/role	Mechanism/definition	Key quantitative data	Experimental basis	Primary citation with DOI/URL and publication date
FKBP-type peptidyl-prolyl cis/trans isomerase (PPIase)	Catalyzes cis/trans isomerization of X-Pro peptide bonds; E. coli SlyD is an FKBP-family PPIase with an N-terminal catalytic domain and a distinct C-terminal metal-binding region	For peptide substrate Suc-Ala-Phe-Pro-Arg-4-nitroanilide, kcat/KM = 29,600 M^-1 s^-1 for full-length SlyD and 18,600 M^-1 s^-1 for the N-terminal fragment; additional peptide specificity constants reported as 6,200 and 5,600 M^-1 s^-1 for other tetrapeptides (burk2007possiblerolesof pages 22-25, erdmann2007thenickel‐regulatedpeptidyl pages 1-5)	Protease-coupled peptide isomerase assays using chromogenic tetrapeptides; truncation analysis of N-terminal catalytic fragment (burk2007possiblerolesof pages 22-25, erdmann2007thenickel‐regulatedpeptidyl pages 1-5)	Hottenrott et al., J. Biol. Chem. (1997-06), DOI: 10.1074/jbc.272.25.15697, https://doi.org/10.1074/jbc.272.25.15697 (erdmann2007thenickel‐regulatedpeptidyl pages 1-5); summarized quantitative values from Burk 2007 excerpt (burk2007possiblerolesof pages 22-25)
Protein-folding catalyst / chaperone-assisted PPIase	SlyD also acts on protein substrates; IF (insert-in-flap) chaperone domain enhances activity on partially folded proteins and supports proline-limited protein folding	For reduced/carboxymethylated RNase T1 refolding, kcat/KM = 6.8 × 10^6 M^-1 s^-1 and KM = 1.65 μM; activity on protein substrates is reported as ~1000-fold higher than on peptide substrates; EcSlyD can accelerate uncatalyzed prolyl isomerization by >10^4-fold (burk2007possiblerolesof pages 22-25, lindemeier2025signalpeptidechaperoning pages 32-35, lindemeier2024positivechargespromote pages 3-4)	Protein refolding kinetics; domain-engineering and deletion studies showing IF-domain dependence for protein, but not small-peptide, substrates (burk2007possiblerolesof pages 22-25, lindemeier2025signalpeptidechaperoning pages 32-35, lindemeier2024positivechargespromote pages 3-4)	Scholz et al., Biochemistry (2006-01), DOI: 10.1021/bi051922n, https://doi.org/10.1021/bi051922n (burk2007possiblerolesof pages 22-25, lindemeier2024positivechargespromote pages 3-4); Lindemeier dissertation summary (2025) citing earlier work (lindemeier2025signalpeptidechaperoning pages 32-35)
Metal-binding, Ni-regulated enzyme	Histidine/cysteine-rich C-terminal tail binds transition metals; Ni2+ binding induces conformational changes that reversibly inhibit the PPIase active site	One excerpt reports 3 metal-binding sites with binding constants 9.5 × 10^5, 4.9 × 10^5, and 4.4 × 10^5 M^-1; addition of 50 μM NiCl2 to 1 μM SlyD caused about 90% decrease in PPIase activity; another study reported binding of 3 Ni2+ ions to the histidine-rich C-terminus (burk2007possiblerolesof pages 22-25, erdmann2007thenickel‐regulatedpeptidyl pages 1-5)	Metal titration/biophysical analysis, CD/NMR structural perturbation, and activity assays in the presence of Ni2+ (burk2007possiblerolesof pages 22-25, kaluarachchi2012slydani pages 59-62, erdmann2007thenickel‐regulatedpeptidyl pages 1-5)	Hottenrott et al., J. Biol. Chem. (1997-06), DOI: 10.1074/jbc.272.25.15697, https://doi.org/10.1074/jbc.272.25.15697 (erdmann2007thenickel‐regulatedpeptidyl pages 1-5); Martino et al., FEBS J. (2009-08), DOI: 10.1111/j.1742-4658.2009.07159.x, https://doi.org/10.1111/j.1742-4658.2009.07159.x (kaluarachchi2012slydani pages 59-62); quantitative constants summarized in Burk 2007 excerpt (burk2007possiblerolesof pages 22-25)
Nickel metallochaperone for hydrogenase maturation	SlyD interacts with HypB and promotes nickel delivery/insertion during [NiFe]-hydrogenase active-site assembly; role is linked more strongly to chaperone/metal-binding functions than to PPIase catalysis	ΔslyD causes a marked reduction in hydrogenase activity; hydrogenase-1 and -2 activities are diminished, HycE processing is reduced, and phenotypes are rescued by excess nickel; SlyD overexpression doubled cellular nickel; estimated abundance ~10^4 molecules/cell (zhang2005arolefor pages 1-1, zhang2005arolefor pages 5-6)	Proteomics identifying HypB-SlyD complex, chemical cross-linking of purified proteins, hydrogenase activity gels, HycE processing assays, and radioactive nickel accumulation assays (zhang2005arolefor pages 1-1, zhang2005arolefor pages 5-6)	Zhang et al., J. Biol. Chem. (2005-02), DOI: 10.1074/jbc.M411799200, https://doi.org/10.1074/jbc.M411799200 (zhang2005arolefor pages 1-1, zhang2005arolefor pages 5-6)
Contribution to cellular nickel accumulation	SlyD increases intracellular nickel availability/retention, consistent with a role in nickel trafficking/homeostasis during anaerobic hydrogenase biogenesis	In ^63Ni uptake assays, ΔslyD accumulated about half as much nickel as wild type; overexpression increased accumulation across tested Ni concentrations (zhang2005arolefor pages 5-6, zhang2005arolefor media 40dc7d88)	Scintillation-based ^63Ni uptake/accumulation experiments; figure evidence shows lower uptake in mutant and increased uptake upon overexpression (zhang2005arolefor pages 5-6, zhang2005arolefor media 40dc7d88)	Zhang et al., J. Biol. Chem. (2005-02), DOI: 10.1074/jbc.M411799200, https://doi.org/10.1074/jbc.M411799200 (zhang2005arolefor pages 5-6, zhang2005arolefor media 40dc7d88)
Tat signal-peptide/substrate chaperone	IF domain recognizes positively charged Tat signal peptides, especially the twin-arginine motif, aided by adjacent hydrophobic residues; arginine is preferred over lysine	HiPIP Tat signal peptide binds SlyD at 1:1 stoichiometry; replacing twin arginines with glutamines reduced affinity by ≥100-fold, with KD = 38.7 ± 2 μM for QQ-HiPIP; single hydrophobic-pocket mutations gave affinities in the 4–10 μM range, whereas double F84N/F96N abolished measurable binding; RR binds more strongly than KK (lindemeier2024positivechargespromote pages 9-11, lindemeier2024positivechargespromote pages 14-16, lindemeier2024positivechargespromote pages 1-3)	Peptide arrays, isothermal titration calorimetry (ITC), and mutational analysis of IF-domain residues (including F84, F96, D101, E108, Y68) (lindemeier2024positivechargespromote pages 9-11, lindemeier2024positivechargespromote pages 14-16, lindemeier2024positivechargespromote pages 1-3, lindemeier2025signalpeptidechaperoning pages 134-137)	Lindemeier et al., PLOS ONE (2024-06), DOI: 10.1371/journal.pone.0305823, https://doi.org/10.1371/journal.pone.0305823 (lindemeier2024positivechargespromote pages 9-11, lindemeier2024positivechargespromote pages 14-16, lindemeier2024positivechargespromote pages 1-3)
Domain architecture relevant to annotation	E. coli SlyD is a three-part cytosolic protein: FKBP catalytic domain, inserted chaperone IF domain, and intrinsically disordered C-terminal nickel-binding tail; IF domain is primary substrate-recognition module for Tat peptides	No single universal numeric value, but IF insertion into FKBP12 increased activity on partially unfolded substrates by 100–200-fold; removal of IF leaves peptide activity largely intact while impairing protein-substrate folding support (lindemeier2025signalpeptidechaperoning pages 32-35, lindemeier2024positivechargespromote pages 3-4)	NMR/crystallographic comparisons, domain swapping, mutational analysis, and substrate-binding studies (lindemeier2025signalpeptidechaperoning pages 32-35, lindemeier2024positivechargespromote pages 3-4)	Kovermann et al., Biol. Chem. (2013-08), DOI: 10.1515/hsz-2013-0137, https://doi.org/10.1515/hsz-2013-0137 (lindemeier2025signalpeptidechaperoning pages 32-35, lindemeier2024positivechargespromote pages 3-4)

Table: This table summarizes experimentally supported functional annotation evidence for E. coli K-12 SlyD (UniProt P0A9K9), emphasizing enzymatic activity, metal binding, hydrogenase maturation, and Tat-signal recognition. It compiles the main quantitative findings and the experimental approaches behind them for rapid use in a research report.

Figure evidence (nickel accumulation phenotype)

Zhang et al. Figure 6 provides direct experimental visualization of reduced 63Ni accumulation in ΔslyD cells and increased accumulation upon SlyD overexpression (zhang2005arolefor media 40dc7d88).

References (URLs and publication dates where available)

Hottenrott S. et al. J Biol Chem. 1997-06. “The E. coli SlyD Is a Metal Ion-regulated Peptidyl-prolyl cis/trans-Isomerase.” https://doi.org/10.1074/jbc.272.25.15697 (erdmann2007thenickel‐regulatedpeptidyl pages 1-5)
Zhang JW. et al. J Biol Chem. 2005-02. “A Role for SlyD in the E. coli Hydrogenase Biosynthetic Pathway.” https://doi.org/10.1074/jbc.M411799200 (zhang2005arolefor pages 1-1)
Martino L. et al. FEBS J. 2009-08. “Interaction of E. coli SlyD with nickel ions…” https://doi.org/10.1111/j.1742-4658.2009.07159.x (kaluarachchi2012slydani pages 59-62)
Kovermann M. et al. Biological Chemistry. 2013-08. “Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD.” https://doi.org/10.1515/hsz-2013-0137 (kaluarachchi2012slydani pages 25-28)
Lindemeier D. et al. PLOS ONE. 2024-06. “Positive charges promote the recognition of proteins by the chaperone SlyD from E. coli.” https://doi.org/10.1371/journal.pone.0305823 (lindemeier2024positivechargespromote pages 1-3)
Han K-Y. et al. Protein Eng Des Sel. 2007-11. “Solubilization… by covalent fusion… SlyD.” https://doi.org/10.1093/protein/gzm055 (han2007solubilizationofaggregationprone pages 1-2)
Andersen KR. et al. Proteins. 2013-08. “LOBSTR eliminates common contaminants from His-tag purification.” https://doi.org/10.1002/prot.24364 (andersen2013optimizede.coliexpressionstrain pages 4-5)

References

(erdmann2007thenickel‐regulatedpeptidyl pages 1-5): Frank Erdmann and Gunter Fischer. The nickel‐regulated peptidyl prolyl cis/trans isomerase slyd. ArXiv, pages 501-518, Mar 2007. URL: https://doi.org/10.1002/9780470028131.ch13, doi:10.1002/9780470028131.ch13. This article has 8 citations.
(zhang2005arolefor pages 1-1): Jie Wei Zhang, Gareth Butland, Jack F. Greenblatt, Andrew Emili, and Deborah B. Zamble. A role for slyd in the escherichia coli hydrogenase biosynthetic pathway*. Journal of Biological Chemistry, 280:4360-4366, Feb 2005. URL: https://doi.org/10.1074/jbc.m411799200, doi:10.1074/jbc.m411799200. This article has 175 citations and is from a domain leading peer-reviewed journal.
(lindemeier2024positivechargespromote pages 1-3): Daniel Lindemeier, Wenke Graubner, Denise Mehner-Breitfeld, Miroslav Malešević, and Thomas Brüser. Positive charges promote the recognition of proteins by the chaperone slyd from escherichia coli. PLOS ONE, 19:e0305823, Jun 2024. URL: https://doi.org/10.1371/journal.pone.0305823, doi:10.1371/journal.pone.0305823. This article has 2 citations and is from a peer-reviewed journal.
(burk2007possiblerolesof pages 22-25): A Burk. Possible roles of slyd in helicobacter pylori hydrogenase maturation. Unknown journal, 2007.
(lindemeier2024positivechargespromote pages 3-4): Daniel Lindemeier, Wenke Graubner, Denise Mehner-Breitfeld, Miroslav Malešević, and Thomas Brüser. Positive charges promote the recognition of proteins by the chaperone slyd from escherichia coli. PLOS ONE, 19:e0305823, Jun 2024. URL: https://doi.org/10.1371/journal.pone.0305823, doi:10.1371/journal.pone.0305823. This article has 2 citations and is from a peer-reviewed journal.
(lindemeier2024positivechargespromote pages 14-16): Daniel Lindemeier, Wenke Graubner, Denise Mehner-Breitfeld, Miroslav Malešević, and Thomas Brüser. Positive charges promote the recognition of proteins by the chaperone slyd from escherichia coli. PLOS ONE, 19:e0305823, Jun 2024. URL: https://doi.org/10.1371/journal.pone.0305823, doi:10.1371/journal.pone.0305823. This article has 2 citations and is from a peer-reviewed journal.
(kaluarachchi2012slydani pages 59-62): H Kaluarachchi. Slyd, a ni (ii) metallochaperone for -hydrogenase biosynthesis in escherichia coli. Unknown journal, 2012.
(zhang2005arolefor pages 5-6): Jie Wei Zhang, Gareth Butland, Jack F. Greenblatt, Andrew Emili, and Deborah B. Zamble. A role for slyd in the escherichia coli hydrogenase biosynthetic pathway*. Journal of Biological Chemistry, 280:4360-4366, Feb 2005. URL: https://doi.org/10.1074/jbc.m411799200, doi:10.1074/jbc.m411799200. This article has 175 citations and is from a domain leading peer-reviewed journal.
(zhang2005arolefor media 40dc7d88): Jie Wei Zhang, Gareth Butland, Jack F. Greenblatt, Andrew Emili, and Deborah B. Zamble. A role for slyd in the escherichia coli hydrogenase biosynthetic pathway*. Journal of Biological Chemistry, 280:4360-4366, Feb 2005. URL: https://doi.org/10.1074/jbc.m411799200, doi:10.1074/jbc.m411799200. This article has 175 citations and is from a domain leading peer-reviewed journal.
(lindemeier2024positivechargespromote pages 9-11): Daniel Lindemeier, Wenke Graubner, Denise Mehner-Breitfeld, Miroslav Malešević, and Thomas Brüser. Positive charges promote the recognition of proteins by the chaperone slyd from escherichia coli. PLOS ONE, 19:e0305823, Jun 2024. URL: https://doi.org/10.1371/journal.pone.0305823, doi:10.1371/journal.pone.0305823. This article has 2 citations and is from a peer-reviewed journal.
(han2007solubilizationofaggregationprone pages 1-2): Kyung-Yeon Han, Jong‐Am Song, K. Ahn, Jin-Seung Park, Hyuk-Seong Seo, and Jeewon Lee. Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive escherichia coli protein, slyd. Protein engineering, design & selection : PEDS, 20 11:543-9, Nov 2007. URL: https://doi.org/10.1093/protein/gzm055, doi:10.1093/protein/gzm055. This article has 52 citations.
(han2007solubilizationofaggregationprone pages 2-3): Kyung-Yeon Han, Jong‐Am Song, K. Ahn, Jin-Seung Park, Hyuk-Seong Seo, and Jeewon Lee. Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive escherichia coli protein, slyd. Protein engineering, design & selection : PEDS, 20 11:543-9, Nov 2007. URL: https://doi.org/10.1093/protein/gzm055, doi:10.1093/protein/gzm055. This article has 52 citations.
(han2007solubilizationofaggregationprone pages 4-6): Kyung-Yeon Han, Jong‐Am Song, K. Ahn, Jin-Seung Park, Hyuk-Seong Seo, and Jeewon Lee. Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive escherichia coli protein, slyd. Protein engineering, design & selection : PEDS, 20 11:543-9, Nov 2007. URL: https://doi.org/10.1093/protein/gzm055, doi:10.1093/protein/gzm055. This article has 52 citations.
(andersen2013optimizede.coliexpressionstrain pages 4-5): Kasper R. Andersen, Nina C. Leksa, and Thomas U. Schwartz. Optimizede. coliexpression strain lobstr eliminates common contaminants from his‐tag purification. Proteins: Structure, Function, and Bioinformatics, 81:1857-1861, Aug 2013. URL: https://doi.org/10.1002/prot.24364, doi:10.1002/prot.24364. This article has 352 citations.
(scholz2006slydproteinsfrom pages 9-10): Christian Scholz, Barbara Eckert, Franz Hagn, Peter Schaarschmidt, Jochen Balbach, and Franz Xaver Schmid. Slyd proteins from different species exhibit high prolyl isomerase and chaperone activities. Biochemistry, 45 1:20-33, Jan 2006. URL: https://doi.org/10.1021/bi051922n, doi:10.1021/bi051922n. This article has 135 citations and is from a peer-reviewed journal.
(lindemeier2025signalpeptidechaperoning pages 32-35): Daniel Lindemeier. Signal peptide chaperoning by slyd and its role in tat transport. Text, Jan 2025. URL: https://doi.org/10.15488/16530, doi:10.15488/16530. This article has 0 citations and is from a peer-reviewed journal.
(lindemeier2025signalpeptidechaperoning pages 134-137): Daniel Lindemeier. Signal peptide chaperoning by slyd and its role in tat transport. Text, Jan 2025. URL: https://doi.org/10.15488/16530, doi:10.15488/16530. This article has 0 citations and is from a peer-reviewed journal.
(kaluarachchi2012slydani pages 25-28): H Kaluarachchi. Slyd, a ni (ii) metallochaperone for -hydrogenase biosynthesis in escherichia coli. Unknown journal, 2012.

Citations

burk2007possiblerolesof pages 22-25
zhang2005arolefor pages 1-1
kaluarachchi2012slydani pages 59-62
zhang2005arolefor pages 5-6
lindemeier2024positivechargespromote pages 1-3
lindemeier2024positivechargespromote pages 9-11
lindemeier2024positivechargespromote pages 14-16
han2007solubilizationofaggregationprone pages 1-2
han2007solubilizationofaggregationprone pages 2-3
han2007solubilizationofaggregationprone pages 4-6
scholz2006slydproteinsfrom pages 9-10
lindemeier2025signalpeptidechaperoning pages 32-35
kaluarachchi2012slydani pages 25-28
lindemeier2024positivechargespromote pages 3-4
lindemeier2025signalpeptidechaperoning pages 134-137
NiFe
https://doi.org/10.1074/jbc.272.25.15697
https://doi.org/10.1021/bi051922n
https://doi.org/10.1111/j.1742-4658.2009.07159.x
https://doi.org/10.1074/jbc.M411799200
https://doi.org/10.1371/journal.pone.0305823
https://doi.org/10.1515/hsz-2013-0137
https://doi.org/10.1093/protein/gzm055
https://doi.org/10.1002/prot.24364
https://doi.org/10.1002/9780470028131.ch13,
https://doi.org/10.1074/jbc.m411799200,
https://doi.org/10.1371/journal.pone.0305823,
https://doi.org/10.1093/protein/gzm055,
https://doi.org/10.1002/prot.24364,
https://doi.org/10.1021/bi051922n,
https://doi.org/10.15488/16530,

Bioreason Rl Review

(SlyD-bioreason-rl-review.md)

BioReason-Pro RL Review: SlyD (E. coli)

Source: SlyD-deep-research-bioreason-rl.md

Correctness: 3/5
Completeness: 2/5

Functional Summary Review

The BioReason functional summary describes SlyD as:

A cytosolic peptidyl-prolyl isomerase that accelerates protein folding by catalyzing cis-trans interconversion at proline bonds and by acting as a soluble folding chaperone. Its FKBP catalytic core drives isomerization, while an auxiliary insertion module enhances affinity for unfolded substrates and supports holdase/chaperone activity. Operating in the bacterial cytoplasm, it collaborates with general proteostasis pathways to stabilize folding intermediates and channel clients toward productive maturation or quality-control pathways.

The PPIase and chaperone aspects are correctly described:
- FKBP-type PPIase activity is accurately captured
- The IF (insert-in-flap) domain's chaperone holdase function is correctly identified
- Cytoplasmic localization is correct
- The dual PPIase + chaperone architecture is well described

However, the summary misses the most distinctive and arguably most important function of SlyD:

Missing nickel metallochaperone function: SlyD's C-terminal histidine/cysteine-rich tail binds up to 7 nickel ions and functions as a nickel metallochaperone in the [NiFe]-hydrogenase maturation pathway. This is a core function assigned GO:0170061 (nickel chaperone activity) in the curated review. The hydrogenase maturation function requires the chaperone domain and C-terminal metal-binding tail but NOT the PPIase activity.
Missing metal-binding properties: The C-terminal tail also binds zinc, copper, and cobalt. Nickel binding reversibly inhibits PPIase activity.
Missing phage biology: SlyD is required for stabilization of the phiX174 lysis protein E, enabling phage-mediated lysis. The protein is literally named "Sensitive to lysis D."

The thinking trace identifies only three InterPro domains (PPIase superfamily, FKBP domain, SlyD-like insertion), presumably because the histidine-rich C-terminal tail is poorly annotated by InterPro. This highlights a limitation: BioReason cannot capture functions not encoded in the InterPro domain annotations.

Comparison with interpro2go:

SlyD has no GO_REF:0000002 annotations in the curated review. BioReason's GO term predictions include peptidyl-prolyl cis-trans isomerase activity (GO:0003755), nickel cation binding (GO:0016151), zinc ion binding (GO:0008270), and cobalt ion binding (GO:0050897). These GO predictions are substantially more informative than the functional summary narrative, which ignores the metal-binding functions entirely. The disconnect between GO predictions and narrative summary is striking.

Notes on thinking trace

The trace correctly identifies the FKBP and SlyD-like insertion domains but does not detect the C-terminal metal-binding tail. It mentions "metal-assisted folding typical of SlyD/SlpA-like proteins" in passing but does not develop this into a functional prediction. The speculative mention of GroEL/GroES as partners is not well supported for SlyD specifically.

📄 View Raw YAML

id: P0A9K9
gene_symbol: SlyD
product_type: PROTEIN
status: IN_PROGRESS
taxon:
  id: NCBITaxon:83333
  label: Escherichia coli (strain K12)
description: SlyD (Sensitive to lysis D) is a multifunctional cytoplasmic protein
  in E. coli that combines three distinct activities in a modular domain architecture.
  The N-terminal FKBP domain harbors peptidyl-prolyl cis-trans isomerase (PPIase)
  activity (EC 5.2.1.8), catalyzing the cis-trans isomerization of Xaa-Pro bonds in
  peptide substrates with high efficiency (kcat/Km approximately 10^6 M-1 s-1 for
  protein substrates; PMID:16388577). An insert-in-flap (IF) domain inserted within
  a loop of the FKBP domain provides a spatially separate chaperone holdase function
  that binds unfolded or partially folded proteins and prevents aggregation (PMID:19356587).
  The C-terminal tail is extremely rich in histidine, cysteine, and acidic residues
  and binds up to 7 nickel ions as well as zinc, copper, and cobalt (PMID:19947632,
  PMID:22047179). SlyD functions as a nickel metallochaperone in the [NiFe]-hydrogenase
  metallocenter assembly pathway, interacting with HypB to stimulate nickel release
  and deliver it for insertion into the hydrogenase large subunit HycE (PMID:15569666,
  PMID:17426034). The hydrogenase maturation function requires the chaperone domain
  and the metal-binding tail but NOT the PPIase activity (PMID:17720786). SlyD PPIase
  activity is reversibly inhibited by nickel binding to the C-terminal region (PMID:9188461,
  PMID:19645725). SlyD is also required for stabilization of the phiX174 lysis protein
  E, enabling phage-mediated lysis (PMID:12100551). SlyD is upregulated under heat
  stress (PMID:17971396).
existing_annotations:
- term:
    id: GO:0003755
    label: peptidyl-prolyl cis-trans isomerase activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: IBA annotation for PPIase activity. SlyD is an FKBP-type PPIase with
      demonstrated catalytic activity for the cis-trans isomerization of Xaa-Pro bonds
      (PMID:9188461, PMID:16388577). The PPIase activity is carried in the N-terminal
      FKBP domain and is conserved across SlyD orthologs.
    action: ACCEPT
    reason: PPIase activity is one of SlyD's two primary enzymatic activities and
      is well-supported by both phylogenetic inference and direct experimental evidence
      (PMID:9188461, PMID:16388577).
    supported_by:
    - reference_id: PMID:9188461
      supporting_text: For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km
        of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment
        were obtained.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: IBA annotation for cytosol localization. Consistent with IDA evidence
      from proteomics studies (PMID:15911532, PMID:18304323) and UniProt subcellular
      location annotation.
    action: ACCEPT
    reason: Cytosol localization is well-established for SlyD by both phylogenetic
      inference and proteomics-based experimental evidence.
- term:
    id: GO:0042026
    label: protein refolding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: IBA annotation for protein refolding. SlyD has both PPIase and chaperone
      activities that together promote protein folding and refolding (PMID:16388577,
      PMID:19356587). The PPIase catalyzes proline-limited refolding steps while the
      IF domain prevents aggregation of folding intermediates.
    action: ACCEPT
    reason: SlyD's combined PPIase and chaperone activities make it a bona fide folding
      helper protein. The protein refolding function is conserved across the SlyD
      family (PMID:16388577) and supported by IDA evidence.
    supported_by:
    - reference_id: PMID:16388577
      supporting_text: All SlyD variants catalyze the proline-limited refolding of
        ribonuclease T1 with very high efficiencies, and the specificity constants
        (kcat/KM) are equal to approximately 10(6) M(-1) s(-1).
- term:
    id: GO:0003755
    label: peptidyl-prolyl cis-trans isomerase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000120
  review:
    summary: IEA annotation for PPIase activity from combined automated methods. Consistent
      with the IBA and IDA annotations for the same term.
    action: ACCEPT
    reason: PPIase activity is a core function supported by multiple evidence types.
      This broader IEA annotation is acceptable.
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: IEA annotation for cytoplasm localization from UniProt subcellular location
      mapping. Broader parent of cytosol. UniProt confirms cytoplasmic localization.
    action: ACCEPT
    reason: Cytoplasmic localization is correct and well-established. The more specific
      cytosol term is annotated with IDA and IBA evidence.
- term:
    id: GO:0016853
    label: isomerase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for isomerase activity from UniProt keyword mapping. SlyD
      is classified as EC 5.2.1.8, which is an isomerase. This is a very broad parent
      of the more specific PPIase annotation.
    action: ACCEPT
    reason: As an EC 5.2.1.8 enzyme, SlyD is indeed an isomerase. This broad IEA is
      acceptable alongside the more specific PPIase annotations.
- term:
    id: GO:0042026
    label: protein refolding
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: IEA annotation for protein refolding from ARBA machine learning models.
      Consistent with the IBA and IDA annotations for the same term.
    action: ACCEPT
    reason: Protein refolding is a well-supported function. This IEA annotation is
      consistent with stronger evidence types.
- term:
    id: GO:0046872
    label: metal ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: IEA annotation for metal ion binding from UniProt keyword mapping. SlyD
      binds multiple metal ions (Ni, Zn, Cu, Co) through its C-terminal histidine/cysteine-rich
      domain (PMID:8300624, PMID:22047179).
    action: ACCEPT
    reason: Metal ion binding is well-established for SlyD. The more specific nickel,
      zinc, copper, and cobalt binding annotations are supported by IDA evidence.
      This broad IEA is acceptable.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:15690043
  review:
    summary: IPI annotation for protein binding from high-throughput interaction study.
      The GOA TSV indicates interaction with HypB (P0AAN3) and GrcA (P68066). SlyD-HypB
      interaction is well-established in the hydrogenase maturation pathway (PMID:15569666,
      PMID:17426034).
    action: MODIFY
    reason: Generic "protein binding" is too vague. The SlyD-HypB interaction is specifically
      in the context of nickel metallochaperone function for hydrogenase maturation.
      GO:0170061 "nickel chaperone activity" better captures the functional significance
      of this interaction. However, for the GrcA interaction, the specific context
      is less clear.
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:16412426
  review:
    summary: IPI annotation for protein binding from Butland et al. (2006) showing
      SlyD-HypB complex formation relevant to hydrogenase biosynthesis.
    action: MODIFY
    reason: This interaction with HypB is specifically in the context of nickel delivery
      for [NiFe]-hydrogenase metallocenter assembly. GO:0170061 "nickel chaperone
      activity" is more informative than generic "protein binding."
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:19402753
  review:
    summary: IPI annotation for protein binding from global interaction atlas of E.
      coli. GOA indicates interaction with HypB (P0AAN3) and GrcA (P68066).
    action: MODIFY
    reason: Same reasoning -- the HypB interaction is related to nickel metallochaperone
      function. GO:0170061 "nickel chaperone activity" is more appropriate.
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:21185288
  review:
    summary: IPI annotation for protein binding from Chung and Zamble (2011) showing
      SlyD interaction with HypB (P0AAN3) and the hydrogenase 3 large subunit HycE
      (P16431). SlyD-HycE interaction is mediated by the chaperone domain and is independent
      of HypB, supporting a direct role in hydrogenase maturation.
    action: MODIFY
    reason: These interactions are specifically in the context of [NiFe]-hydrogenase
      maturation. GO:0170061 "nickel chaperone activity" captures the metallochaperone
      function.
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
    supported_by:
    - reference_id: PMID:21185288
      supporting_text: A SlyD-HycE interaction preceding both iron and nickel insertion
        to the enzyme was detected, mediated by the chaperone domain of SlyD, and
        independent of HypB.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:22016389
  review:
    summary: IPI annotation for protein binding from Tao et al. (2012) showing SlyD
      interaction with HycE (P16431) during nickel insertion to [NiFe] hydrogenase.
    action: MODIFY
    reason: Interaction with HycE is in the context of nickel delivery during hydrogenase
      maturation. GO:0170061 "nickel chaperone activity" is more informative.
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
- term:
    id: GO:0000413
    label: protein peptidyl-prolyl isomerization
  evidence_type: IDA
  original_reference_id: PMID:9188461
  review:
    summary: IDA annotation for protein peptidyl-prolyl isomerization from Hottenrott
      et al. (1997). SlyD was demonstrated to catalyze cis/trans isomerization of
      peptidyl-prolyl bonds with kcat/Km of 29,600 M-1 s-1 for Suc-Ala-Phe-Pro-Arg-4-nitroanilide.
      The PPIase activity is reversibly inhibited by Ni2+ binding to the C-terminal
      histidine-rich region.
    action: ACCEPT
    reason: Direct experimental evidence for the biological process of peptidyl-prolyl
      isomerization. This is one of SlyD's core catalytic activities.
    supported_by:
    - reference_id: PMID:9188461
      supporting_text: the PPIase activity of SlyD is reversibly regulated by binding
        of three Ni2+ ions to the histidine-rich, C-terminal region
- term:
    id: GO:0042026
    label: protein refolding
  evidence_type: IDA
  original_reference_id: PMID:16388577
  review:
    summary: IDA annotation for protein refolding from Scholz et al. (2006). The study
      demonstrated that SlyD proteins from multiple species catalyze proline-limited
      refolding of ribonuclease T1 with very high efficiency and also exhibit pronounced
      chaperone properties that prevent aggregation.
    action: ACCEPT
    reason: Strong experimental evidence for SlyD's role in protein refolding. SlyD
      combines PPIase and chaperone activities that together promote efficient protein
      folding, making it a bona fide folding helper protein.
    supported_by:
    - reference_id: PMID:16388577
      supporting_text: SlyD also exhibits pronounced chaperone properties. Permanently
        unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl
        isomerase activity.
- term:
    id: GO:0051604
    label: protein maturation
  evidence_type: IMP
  original_reference_id: PMID:17426034
  review:
    summary: IMP annotation for protein maturation from Leach et al. (2007). Mutations
      in SlyD that disrupt complex formation with HypB result in deficient hydrogenase
      production in vivo. SlyD stimulates release of nickel from HypB's high-affinity
      Ni(II)-binding site, and a SlyD truncation lacking the C-terminal metal-binding
      tail is not functional in vivo for hydrogenase maturation.
    action: ACCEPT
    reason: Well-supported by mutant phenotype evidence. SlyD is required for proper
      maturation of the [NiFe]-hydrogenase metallocenter. This is a core in vivo function.
    supported_by:
    - reference_id: PMID:17426034
      supporting_text: Mutations in either protein that disrupt complex formation
        in vitro also result in deficient hydrogenase production in vivo, indicating
        that the contact between HypB and SlyD is important for hydrogenase maturation.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:30758762
  review:
    summary: IPI annotation for protein binding from Khorasani-Motlagh et al. (2019)
      showing SlyD-HypB complex formation with KD of 24 nM. SlyD blocks HypA-HypB
      interaction.
    action: MODIFY
    reason: The SlyD-HypB interaction is specifically in the context of nickel delivery
      for [NiFe]-hydrogenase maturation. GO:0170061 "nickel chaperone activity" captures
      the functional significance.
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
    supported_by:
    - reference_id: PMID:30758762
      supporting_text: The affinity of HypB-SlyD was measured by using surface plasmon
        resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide
        and an interaction several fold tighter in the presence of GDP
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:32813023
  review:
    summary: IPI annotation for protein binding from Zhang et al. (2020) showing SlyD
      interaction with Fur (P0A9A9) in the YdiV-SlyD-Fur regulatory pathway for iron
      homeostasis. SlyD's PPIase activity transforms Fur into a form that does not
      bind DNA under iron deficiency conditions.
    action: KEEP_AS_NON_CORE
    reason: This is a genuine protein-protein interaction, but in a non-core context
      (iron homeostasis regulation via YdiV-SlyD-Fur pathway). The "protein binding"
      term is vague, but this interaction represents a secondary regulatory role of
      SlyD rather than its primary functions. The interaction is biologically meaningful
      but peripheral to SlyD's core PPIase/chaperone/metallochaperone functions.
    supported_by:
    - reference_id: PMID:32813023
      supporting_text: High levels of YdiV then transforms Fur into a novel form which
        does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent
        manner.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IDA
  original_reference_id: PMID:15911532
  review:
    summary: IDA annotation for cytosol localization from proteomics study by Corbin
      et al. (2003).
    action: ACCEPT
    reason: Proteomics-based experimental evidence for cytosol localization.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: IDA
  original_reference_id: PMID:18304323
  review:
    summary: IDA annotation for cytosol localization from proteomics study by Ishihama
      et al. (2008).
    action: ACCEPT
    reason: Independent proteomics confirmation of cytosol localization.
- term:
    id: GO:0005507
    label: copper ion binding
  evidence_type: IDA
  original_reference_id: PMID:22047179
  review:
    summary: IDA annotation for copper ion binding from Kaluarachchi et al. (2011).
      The C-terminal domain of SlyD binds Cu(I) with the highest affinity among all
      metals tested. However, in vivo, SlyD has a Ni(II)-specific function.
    action: KEEP_AS_NON_CORE
    reason: Copper binding is experimentally demonstrated in vitro (PMID:22047179),
      but the in vivo function of SlyD is specifically as a nickel metallochaperone.
      The copper binding may not be physiologically relevant, as the same study notes
      "in vivo studies reveal a Ni(II)-specific function for the protein."
    supported_by:
    - reference_id: PMID:22047179
      supporting_text: Although the purified protein is unable to overcome the large
        thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence
        of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein.
- term:
    id: GO:0008270
    label: zinc ion binding
  evidence_type: IDA
  original_reference_id: PMID:22047179
  review:
    summary: IDA annotation for zinc ion binding from Kaluarachchi et al. (2011).
      SlyD binds zinc with affinity similar to nickel in vitro, but in vivo function
      is nickel-specific.
    action: KEEP_AS_NON_CORE
    reason: Zinc binding is experimentally demonstrated (PMID:22047179, PMID:8300624),
      but SlyD's in vivo metallochaperone function is specific to nickel. Zinc binding
      may represent a property of the metal-binding domain rather than a specific
      biological function.
    supported_by:
    - reference_id: PMID:22047179
      supporting_text: 'The order of affinities of SlyD for the metals examined is
        as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I)'
- term:
    id: GO:0016151
    label: nickel cation binding
  evidence_type: IDA
  original_reference_id: PMID:22047179
  review:
    summary: IDA annotation for nickel cation binding from Kaluarachchi et al. (2011).
      SlyD binds multiple Ni(II) ions with submicromolar affinity in the C-terminal
      domain.
    action: ACCEPT
    reason: Nickel binding is the physiologically relevant metal-binding function
      of SlyD, directly related to its role as a nickel metallochaperone for [NiFe]-hydrogenase
      maturation (PMID:22047179, PMID:19947632).
    supported_by:
    - reference_id: PMID:22047179
      supporting_text: in vivo studies reveal a Ni(II)-specific function for the protein.
- term:
    id: GO:0050897
    label: cobalt ion binding
  evidence_type: IDA
  original_reference_id: PMID:22047179
  review:
    summary: IDA annotation for cobalt ion binding from Kaluarachchi et al. (2011).
      SlyD binds Co(II) with appreciable affinity but lower than Ni(II) or Zn(II).
    action: KEEP_AS_NON_CORE
    reason: Cobalt binding is experimentally demonstrated in vitro but likely not
      physiologically relevant. In vivo, SlyD functions specifically in nickel homeostasis
      (PMID:22047179).
- term:
    id: GO:0008270
    label: zinc ion binding
  evidence_type: IDA
  original_reference_id: PMID:8300624
  review:
    summary: IDA annotation for zinc ion binding from Wuelfing et al. (1994). The
      original characterization of SlyD showed it binds Ni2+ and Zn2+ tightly with
      1:1 stoichiometry.
    action: KEEP_AS_NON_CORE
    reason: Zinc binding is experimentally demonstrated but likely not the primary
      in vivo function. Same reasoning as the other zinc binding annotation.
    supported_by:
    - reference_id: PMID:8300624
      supporting_text: The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry,
        Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly
        at all.
- term:
    id: GO:0016151
    label: nickel cation binding
  evidence_type: IDA
  original_reference_id: PMID:19645725
  review:
    summary: IDA annotation for nickel cation binding from Martino et al. (2009).
      NMR studies showed nickel binding causes conformational rearrangements in the
      PPIase domain, modulating isomerase activity.
    action: ACCEPT
    reason: Nickel binding is well-established and directly relevant to regulation
      of SlyD's PPIase activity and its metallochaperone function.
    supported_by:
    - reference_id: PMID:19645725
      supporting_text: the interaction of SlyD with nickel ions entails participation
        of the novel structural features of the PPIase domain, eliciting structural
        alterations of the catalytic pocket
- term:
    id: GO:0016151
    label: nickel cation binding
  evidence_type: IDA
  original_reference_id: PMID:19947632
  review:
    summary: IDA annotation for nickel cation binding from Kaluarachchi et al. (2009).
      ESI-MS showed SlyD binds up to 7 nickel ions in a noncooperative manner with
      submicromolar affinity. Cysteine residues in the C-terminal domain confer tighter
      affinity and increased binding capacity.
    action: ACCEPT
    reason: Detailed characterization of SlyD's nickel binding properties directly
      related to its metallochaperone function.
    supported_by:
    - reference_id: PMID:19947632
      supporting_text: SlyD binds up to seven nickel ions in a noncooperative manner
        with submicromolar affinity (<2 microM, upper limit)
- term:
    id: GO:0016151
    label: nickel cation binding
  evidence_type: IDA
  original_reference_id: PMID:8300624
  review:
    summary: IDA annotation for nickel cation binding from Wuelfing et al. (1994).
      The original characterization showed tight Ni2+ binding.
    action: ACCEPT
    reason: The first report of SlyD's nickel binding activity, consistent with subsequent
      studies showing up to 7 Ni ions bound.
    supported_by:
    - reference_id: PMID:8300624
      supporting_text: The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry
- term:
    id: GO:0051082
    label: unfolded protein binding
  evidence_type: IDA
  original_reference_id: PMID:19356587
  review:
    summary: IDA annotation for unfolded protein binding from Weininger et al. (2009).
      NMR titration experiments revealed that the IF domain recognizes and binds unfolded
      or partially folded proteins and peptides. Insulin aggregation is markedly slowed
      by SlyD. However, this binding is in the context of SlyD's chaperone holdase
      activity, which prevents aggregation and promotes folding.
    action: MODIFY
    reason: SlyD's IF domain functions as a genuine chaperone holdase that binds unfolded
      proteins, prevents aggregation, and promotes folding (PMID:19356587, PMID:16388577).
      The term GO:0051082 "unfolded protein binding" describes only the binding aspect.
      GO:0140309 "unfolded protein carrier activity" better captures the holdase function
      that prevents aggregation and escorts unfolded substrates. Unlike RidA or CpxP,
      SlyD's chaperone function is constitutive and a core activity of the IF domain.
    proposed_replacement_terms:
    - id: GO:0051082
      label: unfolded protein binding (retain until holdase NTR is created)
    supported_by:
    - reference_id: PMID:19356587
      supporting_text: NMR titration experiments revealed that the IF domain recognizes
        and binds unfolded or partially folded proteins and peptides. Insulin aggregation
        is markedly slowed by SlyD* as evidenced by two-dimensional NMR spectroscopy
        in real time, probably due to SlyD* binding to denatured insulin.
- term:
    id: GO:0003755
    label: peptidyl-prolyl cis-trans isomerase activity
  evidence_type: IMP
  original_reference_id: PMID:17720786
  review:
    summary: IMP annotation for PPIase activity from Zhang et al. (2007). Mutations
      that decrease PPIase activity (I42S, F132Y) do not produce corresponding decreases
      in hydrogenase production, demonstrating that PPIase activity is separable from
      the metallochaperone function.
    action: ACCEPT
    reason: This IMP evidence supports the existence of PPIase activity in SlyD, even
      though the study's main finding is that PPIase is NOT required for hydrogenase
      maturation. The mutant phenotype evidence confirms PPIase is a real enzymatic
      activity of SlyD.
    supported_by:
    - reference_id: PMID:17720786
      supporting_text: Mutations that result in deficient PPIase activity do not produce
        corresponding decreases in the other activities of SlyD in vitro or in hydrogenase
        production levels in vivo.
- term:
    id: GO:0050821
    label: protein stabilization
  evidence_type: IMP
  original_reference_id: PMID:17971396
  review:
    summary: IMP annotation for protein stabilization from Han et al. (2007). SlyD
      was effective as a fusion partner in preventing aggregation and increasing solubility
      of aggregation-prone heterologous proteins. SlyD sequesters interactive surfaces
      of heterologous proteins associated with inclusion body formation.
    action: KEEP_AS_NON_CORE
    reason: The protein stabilization function is demonstrated in the context of a
      biotechnological fusion partner system, which reflects SlyD's chaperone properties.
      This is a real activity but the in vivo significance as a standalone "protein
      stabilization" function in E. coli is less clear than its PPIase and metallochaperone
      roles.
    supported_by:
    - reference_id: PMID:17971396
      supporting_text: SlyD was very effective in sequestering interactive surfaces
        of heterologous proteins associated with non-specific protein-protein interactions
        and the formation of inclusion bodies
- term:
    id: GO:0009408
    label: response to heat
  evidence_type: IEP
  original_reference_id: PMID:17971396
  review:
    summary: IEP annotation for response to heat from Han et al. (2007). SlyD synthesis
      increased 3.37-fold under heat shock conditions compared to non-stress conditions,
      even when 29 out of 858 soluble proteins were eliminated by the stress.
    action: KEEP_AS_NON_CORE
    reason: SlyD upregulation under heat stress is supported by expression profiling,
      but this is an IEP annotation (expression pattern) rather than functional evidence.
      The heat stress response may reflect SlyD's general chaperone properties but
      is not its primary function.
    supported_by:
    - reference_id: PMID:17971396
      supporting_text: a 3.37-fold increase induced by heat shock treatment was observed
        in the synthesis level of SlyD compared with a non-stress condition
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:15569666
  review:
    summary: IPI annotation for protein binding from Zhang et al. (2005) showing SlyD-HypB
      interaction. Sequential peptide affinity purification revealed SlyD in complex
      with HypB. Deletion of slyD resulted in reduced hydrogenase activity and diminished
      nickel accumulation.
    action: MODIFY
    reason: The SlyD-HypB interaction is specifically in the context of nickel delivery
      for [NiFe]-hydrogenase metallocenter assembly. GO:0170061 "nickel chaperone
      activity" is more informative than generic "protein binding."
    proposed_replacement_terms:
    - id: GO:0170061
      label: nickel chaperone activity
    supported_by:
    - reference_id: PMID:15569666
      supporting_text: Deletion of the slyD gene resulted in a marked reduction of
        the hydrogenase activity in cell extracts
- term:
    id: GO:0003755
    label: peptidyl-prolyl cis-trans isomerase activity
  evidence_type: IDA
  original_reference_id: PMID:9188461
  review:
    summary: IDA annotation for PPIase activity from Hottenrott et al. (1997). Directly
      measured catalytic activity with kcat/Km of 29,600 M-1 s-1 for Suc-Ala-Phe-Pro-Arg-4-nitroanilide
      substrate. PPIase activity is of the FKBP type and is reversibly regulated by
      Ni2+ binding.
    action: ACCEPT
    reason: Direct enzymatic assay demonstrating PPIase catalytic activity. This is
      the definitive experimental evidence for SlyD's PPIase function.
    supported_by:
    - reference_id: PMID:9188461
      supporting_text: Only when using trypsin instead of chymotrypsin as helper enzyme
        in the PPIase assay, the enzymatic activity of full-length SlyD and its N-terminal
        fragment can be measured.
- term:
    id: GO:0170061
    label: nickel chaperone activity
  evidence_type: IDA
  original_reference_id: PMID:15569666
  review:
    summary: NEW annotation for nickel chaperone activity. SlyD functions as a nickel
      metallochaperone that participates in the nickel insertion step of [NiFe]-hydrogenase
      metallocenter assembly. Deletion of slyD results in reduced hydrogenase activity,
      which is rescued by excess nickel but not zinc (PMID:15569666). SlyD interacts
      with HypB and stimulates nickel release from HypB's high-affinity Ni(II)-binding
      site (PMID:17426034). SlyD also directly interacts with HycE, the hydrogenase
      3 large subunit (PMID:21185288).
    action: NEW
    reason: Nickel metallochaperone activity is one of SlyD's most well-characterized
      and biologically important functions. GO:0170061 "nickel chaperone activity"
      (defined as "Directly binding to and delivering nickel ions to a target protein")
      precisely describes SlyD's role in delivering nickel to [NiFe]-hydrogenase.
      This function is supported by multiple studies (PMID:15569666, PMID:17426034,
      PMID:21185288, PMID:22047179) and requires the chaperone domain and C-terminal
      metal-binding tail but not the PPIase activity (PMID:17720786).
    supported_by:
    - reference_id: PMID:15569666
      supporting_text: These experiments demonstrate that SlyD has a role in the nickel
        insertion step of the hydrogenase maturation pathway
    - reference_id: PMID:17426034
      supporting_text: SlyD stimulates release of nickel from the high affinity Ni(II)-binding
        site of HypB, an activity that is also disrupted by mutations that affect
        complex formation.
    additional_reference_ids:
    - PMID:17426034
    - PMID:21185288
    - PMID:22047179
references:
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000043
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
    vocabulary mapping, accompanied by conservative changes to GO terms applied by
    UniProt
  findings: []
- id: GO_REF:0000117
  title: Electronic Gene Ontology annotations created by ARBA machine learning models
  findings: []
- id: GO_REF:0000120
  title: Combined Automated Annotation using Multiple IEA Methods
  findings: []
- id: PMID:8300624
  title: An Escherichia coli protein consisting of a domain homologous to FK506-binding
    proteins (FKBP) and a new metal binding motif.
  findings:
  - statement: SlyD binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry
    supporting_text: The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry,
      Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly
      at all.
  - statement: SlyD consists of an N-terminal FKBP domain and a C-terminal metal-binding
      domain
    supporting_text: It consists of two domains, of which the first (146 amino acids)
      shows some homology to the FK506-binding proteins. The second domain (50 amino
      acids) is extremely rich in potentially metal-binding amino acids, such as histidine,
      cysteine, and acidic amino acids.
- id: PMID:9188461
  title: The Escherichia coli SlyD is a metal ion-regulated peptidyl-prolyl cis/trans-isomerase.
  findings:
  - statement: SlyD is an FKBP-type PPIase with kcat/Km of 29,600 M-1 s-1
    supporting_text: For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km
      of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment were
      obtained.
  - statement: SlyD PPIase activity is reversibly inhibited by nickel binding
    supporting_text: the PPIase activity of SlyD is reversibly regulated by binding
      of three Ni2+ ions to the histidine-rich, C-terminal region
- id: PMID:12100551
  title: The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization
    of the E lysis protein of bacteriophage phi X174.
  findings:
  - statement: SlyD stabilizes the phiX174 lysis protein E
- id: PMID:15569666
  title: A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway.
  findings:
  - statement: SlyD interacts with HypB in the hydrogenase biosynthetic pathway
    supporting_text: The analysis of the proteins in a complex with HypB revealed
      the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has
      not been previously linked to the hydrogenase biosynthetic pathway.
  - statement: Deletion of slyD results in reduced hydrogenase activity rescuable
      by nickel
    supporting_text: Deletion of the slyD gene resulted in a marked reduction of the
      hydrogenase activity in cell extracts prepared from anaerobic cultures
- id: PMID:15690043
  title: Interaction network containing conserved and essential protein complexes
    in Escherichia coli.
  findings: []
- id: PMID:15911532
  title: Localization, annotation, and comparison of the Escherichia coli K-12 proteome
    under two states of growth.
  findings: []
- id: PMID:16388577
  title: SlyD proteins from different species exhibit high prolyl isomerase and chaperone
    activities.
  findings:
  - statement: SlyD catalyzes proline-limited refolding with very high efficiency
    supporting_text: All SlyD variants catalyze the proline-limited refolding of ribonuclease
      T1 with very high efficiencies, and the specificity constants (kcat/KM) are
      equal to approximately 10(6) M(-1) s(-1).
  - statement: SlyD exhibits pronounced chaperone properties for unfolded proteins
    supporting_text: SlyD also exhibits pronounced chaperone properties. Permanently
      unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl
      isomerase activity.
- id: PMID:16412426
  title: 'Interactions of the Escherichia coli hydrogenase biosynthetic proteins:
    HybG complex formation.'
  findings: []
- id: PMID:17426034
  title: The role of complex formation between the Escherichia coli hydrogenase accessory
    factors HypB and SlyD.
  findings:
  - statement: SlyD stimulates nickel release from HypB for hydrogenase maturation
    supporting_text: SlyD stimulates release of nickel from the high affinity Ni(II)-binding
      site of HypB, an activity that is also disrupted by mutations that affect complex
      formation.
  - statement: SlyD C-terminal metal-binding tail is required for in vivo hydrogenase
      function
    supporting_text: a SlyD truncation lacking the C-terminal metal-binding tail still
      interacts with HypB but is deficient in stimulating metal release and is not
      functional in vivo
- id: PMID:17720786
  title: The peptidyl-prolyl isomerase activity of SlyD is not required for maturation
    of Escherichia coli hydrogenase.
  findings:
  - statement: PPIase activity of SlyD is not required for hydrogenase maturation
    supporting_text: Mutations that result in deficient PPIase activity do not produce
      corresponding decreases in the other activities of SlyD in vitro or in hydrogenase
      production levels in vivo.
- id: PMID:17971396
  title: Solubilization of aggregation-prone heterologous proteins by covalent fusion
    of stress-responsive Escherichia coli protein, SlyD.
  findings:
  - statement: SlyD is upregulated 3.37-fold under heat shock stress
    supporting_text: a 3.37-fold increase induced by heat shock treatment was observed
      in the synthesis level of SlyD compared with a non-stress condition
  - statement: SlyD facilitates folding and increases solubility of aggregation-prone
      proteins
    supporting_text: SlyD was very effective in sequestering interactive surfaces
      of heterologous proteins associated with non-specific protein-protein interactions
      and the formation of inclusion bodies
- id: PMID:18304323
  title: Protein abundance profiling of the Escherichia coli cytosol.
  findings: []
- id: PMID:19356587
  title: 'NMR solution structure of SlyD from Escherichia coli: spatial separation
    of prolyl isomerase and chaperone function.'
  findings:
  - statement: SlyD IF domain recognizes and binds unfolded proteins
    supporting_text: NMR titration experiments revealed that the IF domain recognizes
      and binds unfolded or partially folded proteins and peptides.
  - statement: SlyD prevents insulin aggregation via chaperone holdase function
    supporting_text: Insulin aggregation is markedly slowed by SlyD* as evidenced
      by two-dimensional NMR spectroscopy in real time, probably due to SlyD* binding
      to denatured insulin.
- id: PMID:19402753
  title: Global functional atlas of Escherichia coli encompassing previously uncharacterized
    proteins.
  findings: []
- id: PMID:19645725
  title: The interaction of the Escherichia coli protein SlyD with nickel ions illuminates
    the mechanism of regulation of its peptidyl-prolyl isomerase activity.
  findings:
  - statement: Nickel binding causes conformational changes in PPIase domain regulating
      activity
    supporting_text: the interaction of SlyD with nickel ions entails participation
      of the novel structural features of the PPIase domain, eliciting structural
      alterations of the catalytic pocket
- id: PMID:19947632
  title: The Ni(II)-binding properties of the metallochaperone SlyD.
  findings:
  - statement: SlyD binds up to 7 nickel ions noncooperatively with submicromolar
      affinity
    supporting_text: SlyD binds up to seven nickel ions in a noncooperative manner
      with submicromolar affinity (<2 microM, upper limit)
  - statement: Cysteine residues in C-terminal domain confer tighter Ni affinity
    supporting_text: the characterization of SlyD mutants demonstrates that the cysteine
      residues in the C-terminal domain confer tighter affinity as well as increased
      binding capacity to SlyD
- id: PMID:21185288
  title: The Escherichia coli metal-binding chaperone SlyD interacts with the large
    subunit of [NiFe]-hydrogenase 3.
  findings:
  - statement: SlyD directly interacts with HycE via its chaperone domain
    supporting_text: A SlyD-HycE interaction preceding both iron and nickel insertion
      to the enzyme was detected, mediated by the chaperone domain of SlyD, and independent
      of HypB.
- id: PMID:22016389
  title: Protein interactions and localization of the Escherichia coli accessory protein
    HypA during nickel insertion to [NiFe] hydrogenase.
  findings: []
- id: PMID:22047179
  title: Metal selectivity of the Escherichia coli nickel metallochaperone, SlyD.
  findings:
  - statement: SlyD binds Ni, Zn, Cu, Co but has Ni-specific in vivo function
    supporting_text: Although the purified protein is unable to overcome the large
      thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence
      of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein.
  - statement: Metal affinity order is Mn,Fe < Co < Ni ~ Zn << Cu
    supporting_text: 'The order of affinities of SlyD for the metals examined is as
      follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I)'
- id: PMID:30758762
  title: Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel
    maturation factors.
  findings:
  - statement: SlyD-HypB complex has KD of 24 nM
    supporting_text: The affinity of HypB-SlyD was measured by using surface plasmon
      resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and
      an interaction several fold tighter in the presence of GDP
  - statement: SlyD blocks HypA-HypB interaction
    supporting_text: A ternary complex between all three proteins was not detected,
      and instead SlyD blocked the interaction of HypA with HypB in competitive binding
      experiments.
- id: PMID:32813023
  title: YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding
    ability of Fur in a SlyD-dependent manner.
  findings:
  - statement: SlyD participates in iron homeostasis regulation via YdiV-SlyD-Fur
      pathway
    supporting_text: High levels of YdiV then transforms Fur into a novel form which
      does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner.
core_functions:
- molecular_function:
    id: GO:0003755
    label: peptidyl-prolyl cis-trans isomerase activity
  directly_involved_in:
  - id: GO:0042026
    label: protein refolding
  locations:
  - id: GO:0005829
    label: cytosol
  description: SlyD is an FKBP-type PPIase that catalyzes the cis-trans isomerization
    of Xaa-Pro peptide bonds with high efficiency (kcat/Km approximately 10^6 M-1
    s-1 for protein substrates). The PPIase activity is carried in the N-terminal
    FKBP domain and is reversibly inhibited by Ni2+ binding to the C-terminal region.
    Together with the chaperone holdase function of the IF domain, the PPIase accelerates
    slow steps of protein folding.
  supported_by:
  - reference_id: PMID:9188461
    supporting_text: For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km
      of 29,600 M-1 s-1 for SlyD
  - reference_id: PMID:16388577
    supporting_text: All SlyD variants catalyze the proline-limited refolding of ribonuclease
      T1 with very high efficiencies
- molecular_function:
    id: GO:0170061
    label: nickel chaperone activity
  directly_involved_in:
  - id: GO:0051604
    label: protein maturation
  locations:
  - id: GO:0005829
    label: cytosol
  description: SlyD functions as a nickel metallochaperone in the [NiFe]-hydrogenase
    maturation pathway. It binds up to 7 Ni(II) ions in its C-terminal histidine/cysteine-rich
    domain, interacts with HypB to stimulate nickel release, and directly interacts
    with the hydrogenase large subunit HycE to deliver nickel for metallocenter assembly.
    This function requires the chaperone domain and C-terminal metal-binding tail
    but NOT the PPIase activity.
  supported_by:
  - reference_id: PMID:15569666
    supporting_text: These experiments demonstrate that SlyD has a role in the nickel
      insertion step of the hydrogenase maturation pathway
  - reference_id: PMID:17426034
    supporting_text: SlyD stimulates release of nickel from the high affinity Ni(II)-binding
      site of HypB
- molecular_function:
    id: GO:0051082
    label: unfolded protein binding
  directly_involved_in:
  - id: GO:0042026
    label: protein refolding
  locations:
  - id: GO:0005829
    label: cytosol
  description: The insert-in-flap (IF) domain of SlyD provides chaperone holdase activity
    that binds unfolded or partially folded proteins, prevents their aggregation,
    and promotes correct folding. The chaperone activity is spatially separate from
    the PPIase active site. The IF domain also mediates SlyD's interaction with hydrogenase
    substrates and Tat signal sequences.
  supported_by:
  - reference_id: PMID:19356587
    supporting_text: NMR titration experiments revealed that the IF domain recognizes
      and binds unfolded or partially folded proteins and peptides
  - reference_id: PMID:16388577
    supporting_text: SlyD also exhibits pronounced chaperone properties. Permanently
      unfolded proteins bind with high affinity to SlyD

SlyD

Gene Description

Existing Annotations Review

Core Functions

References

📚 Additional Documentation

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Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Output

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Research report: Escherichia coli K-12 slyD (UniProt P0A9K9) — functional annotation

0) Target verification (critical identity check)

1) Key concepts and current understanding (definitions and molecular function)

1.1 Peptidyl-prolyl cis/trans isomerase (PPIase) activity

1.2 Chaperone / folding-catalyst behavior (IF domain)

1.3 Metal binding and metal-dependent regulation

2) Biological role(s), pathways, and cellular location

2.1 [NiFe]-hydrogenase maturation and nickel trafficking (cytosolic maturation pathway)

2.2 Tat signal peptide/substrate recognition (2024 development)

3) Recent developments and latest research (prioritizing 2023–2024)

4) Current applications and real-world implementations

4.1 SlyD as a solubility/folding fusion partner in recombinant expression

4.2 SlyD as a major contaminant in His-tag metal affinity purification (IMAC)

4.3 Use of SlyD* fusions in diagnostic immunoassays

5) Expert opinion and synthesis (authoritative analysis)

5.1 Multi-function protein with separable but coupled modules

5.2 Mechanistic plausibility for coupling nickel biology to folding control

5.3 Emergent chaperone role in Tat pathway substrate handling (2024)

6) Key quantitative statistics and data points (selected)

Evidence summary table

Figure evidence (nickel accumulation phenotype)

References (URLs and publication dates where available)

Citations

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