Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0030288 outer membrane-bounded periplasmic space	IEA GO_REF:0000117	ACCEPT	Summary: FliY is a periplasmic solute-binding protein in Gram-negative bacteria. It contains an N-terminal signal peptide (residues 1-28) directing export across the inner membrane into the periplasm. As a component of the FliY-YecSC ABC transporter, it operates as a soluble protein in the outer membrane-bounded periplasmic space, where it captures L-cystine and delivers it to the inner-membrane YecSC complex. This localization is well-supported by the protein's signal peptide, its membership in the bacterial solute-binding protein 3 family, and the conserved architecture of Gram-negative ABC import systems. The ARBA prediction is consistent with the known biology.
GO:0030313 cell envelope	IEA GO_REF:0000044	KEEP AS NON CORE	Summary: The cell envelope annotation is derived from UniProtKB-SubCell mapping and is not incorrect -- FliY does reside within the cell envelope. However, this is a very broad term (GO:0030313) that encompasses the outer membrane, periplasmic space, inner membrane, and cell wall. The more specific annotation to outer membrane-bounded periplasmic space (GO:0030288) already captures the precise localization of FliY. Retaining this annotation as non-core since it is accurate but less informative than the periplasmic space annotation.
GO:0016597 amino acid binding	ISS file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md	NEW	Summary: FliY is the periplasmic substrate-binding protein of the FliY-YecSC (TcyJLN) ABC transporter. Its primary molecular function is binding L-cystine with high affinity in the periplasm. The amino acid binding term captures this substrate-binding role. This is inferred from sequence similarity to the well-characterized E. coli FliY ortholog and from membership in the bacterial solute-binding protein family 3. The CDD annotation (cd13711, PBP2_Ngo0372_TcyA) further supports this functional assignment. Supporting Evidence: file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md [Inferred from E. coli ortholog] FliY serves as the periplasmic substrate-binding component of an ATP-binding cassette (ABC) transporter complex designated FliY-YecSC. This system functions as a high-affinity importer of L-cystine.
GO:0015811 L-cystine transport	ISS file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md	NEW	Summary: FliY functions as the substrate-capture component of the cystine ABC transporter system. Its role in L-cystine transport is inferred from sequence similarity to the E. coli FliY ortholog, which has been experimentally demonstrated to be essential for cystine import via the FliY-YecSC system. The UniProt submission name ("Cysteine transport protein") and CDD domain hit (PBP2_Ngo0372_TcyA) are consistent with this functional assignment. Supporting Evidence: file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md [Inferred from E. coli ortholog] Genetic deletion studies definitively established that FliY, together with a second cystine transporter YdjN (TcyP), constitutes one of only two cystine import systems in the bacterium. Double mutants lacking both fliY and ydjN are completely unable to import detectable levels of cystine.
GO:0043190 ATP-binding cassette (ABC) transporter complex	ISS file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md	NEW	Summary: FliY is the periplasmic binding protein subunit of the FliY-YecSC ABC transporter complex. This complex membership is inferred from homology to the E. coli system where FliY, YecS, and YecC form the three-component ABC transporter, and from the UniProt annotation as an ABC superfamily periplasmic binding protein. Supporting Evidence: file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md [Inferred from E. coli ortholog] The FliY-YecSC system operates through the canonical ABC transporter mechanism involving three protein components: FliY is the periplasmic substrate-binding protein, YecS the ATPase component, and YecC the integral membrane permease.

Core Functions

FliY is the periplasmic substrate-binding protein component of the FliY-YecSC (TcyJLN) ABC transporter complex. It captures L-cystine in the periplasm with high affinity and delivers it to the membrane-associated transporter components for ATP-dependent import. This supports sulfur assimilation under conditions of sulfur limitation.

Molecular Function:

amino acid binding

Directly Involved In:

L-cystine transport

Cellular Locations:

outer membrane-bounded periplasmic space

In Complex:

ATP-binding cassette (ABC) transporter complex

Supporting Evidence:

file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md
[Inferred from E. coli ortholog characterization] FliY serves as the periplasmic substrate-binding component of an ATP-binding cassette (ABC) transporter complex designated FliY-YecSC (also known as TcyJLN). This system functions as a high-affinity importer of L-cystine. The FliY-YecSC system achieves half-maximal transport at approximately 30 nanomolar cystine concentration.

References

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

PMID:22125637

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes

This is the genome sequencing paper for Xenorhabdus nematophila strain ATCC 19061, which identified the fliY gene (locus tag XNC1_0570) encoding the cysteine transport protein.

file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md

Deep research report on D3VIU4 fliY from Xenorhabdus nematophila

FliY serves as the periplasmic substrate-binding component of the FliY-YecSC (TcyJLN) ABC transporter complex for high-affinity L-cystine import. Studies in E. coli demonstrate that the FliY-YecSC system achieves half-maximal transport at approximately 30 nM cystine, about 100-fold higher affinity than the alternative YdjN transporter. The protein belongs to bacterial solute-binding protein family 3 and operates in the periplasmic space of Gram-negative bacteria.

Deep Research

Falcon

(D3VIU4-deep-research-falcon.md)

Comprehensive Research Report: Gene fliY (D3VIU4) from Xenorhabdus nematophila Falcon Edison Scientific Literature 7 citations 1 artifacts 2026-06-18T18:26:07.737234

Edison artifact artifact-00 (text/markdown)

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.

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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.

Comprehensive Research Report: Gene fliY (D3VIU4) from Xenorhabdus nematophila

Executive Summary

The fliY gene (UniProt: D3VIU4, locus tag: XNC1_0570) from Xenorhabdus nematophila strain ATCC 19061 encodes a periplasmic substrate-binding protein that functions as the substrate-capture component of a high-affinity ABC transporter system for cystine/cysteine uptake. While direct experimental characterization of this specific gene from Xenorhabdus nematophila has not been reported in the literature, extensive functional studies of orthologous proteins in closely related enterobacteria, particularly Escherichia coli, provide a robust foundation for inferring its function. The protein belongs to the bacterial solute-binding protein family 3 and plays a critical role in sulfur assimilation under conditions of sulfur limitation.

Protein Function and Molecular Mechanism

Primary Function: High-Affinity Cystine Transport

The FliY protein serves as the periplasmic substrate-binding component of an ATP-binding cassette (ABC) transporter complex designated FliY-YecSC (also known as TcyJLN in alternative nomenclature) (imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 5-7). This system functions as a high-affinity importer of L-cystine, the oxidized dimeric form of cysteine that predominates in aerobic extracellular environments (zhou2020escherichiacolik12 pages 1-2, imlay2015physiologicalrolesand pages 1-2).

In E. coli, genetic deletion studies definitively established that FliY, together with a second cystine transporter YdjN (TcyP), constitutes one of only two cystine import systems in the bacterium. Double mutants lacking both fliY and ydjN are completely unable to import detectable levels of cystine and cannot grow when cystine is provided as the sole sulfur source (imlay2015physiologicalrolesand pages 4-5, imlay2015physiologicalrolesand pages 5-7). This demonstrates the essential and non-redundant role of the FliY-YecSC system in cystine acquisition.

Transport Mechanism and Molecular Partners

The FliY-YecSC system operates through the canonical ABC transporter mechanism involving three protein components (imlay2015physiologicalrolesand pages 5-7):

FliY: The periplasmic substrate-binding protein that captures cystine from the extracellular milieu with high affinity
YecS: The ATPase component that provides energy for transport through ATP hydrolysis
YecC: The integral membrane permease that forms the translocation channel

FliY is synthesized with a signal sequence that directs its secretion across the inner membrane into the periplasmic space, where it functions as a soluble protein to bind substrate and deliver it to the membrane-associated YecSC complex (imlay2015physiologicalrolesand pages 5-7, imlay2015physiologicalrolesand pages 7-9). Genetic complementation studies confirmed that deletion of yecS phenocopies deletion of fliY, establishing that YecS and YecC are the cognate membrane partners of FliY (imlay2015physiologicalrolesand pages 5-7).

Substrate Specificity and Binding Affinity

The FliY-YecSC system exhibits remarkably high affinity for its substrate. Transport kinetic studies in E. coli determined that the system achieves half-maximal transport at approximately 30 nanomolar cystine concentration (imlay2015physiologicalrolesand pages 9-10). This represents approximately 100-fold higher affinity compared to the alternative cystine transporter YdjN (Km ~2 μM), positioning FliY-YecSC as the primary system for cystine acquisition when substrate availability is limited (imlay2015physiologicalrolesand pages 9-10).

While L-cystine is the primary physiological substrate, the FliY-YecSC system also demonstrates ability to transport diaminopimelate (DAP), indicating some promiscuity for structurally related diamino compounds (imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 9-10). However, cystine remains the key substrate for sulfur acquisition under natural growth conditions.

Competitive growth experiments at very low cell densities demonstrated that the FliY system is essential for bacterial fitness when cystine concentrations are in the nanomolar range. Wild-type cells successfully outgrew fliY deletion mutants under these limiting conditions, whereas both strains grew comparably when micromolar cystine was provided (allowing the lower-affinity YdjN transporter to compensate) (imlay2015physiologicalrolesand pages 9-10).

Cellular Localization

FliY functions as a soluble periplasmic binding protein in Gram-negative bacteria (imlay2015physiologicalrolesand pages 5-7, imlay2015physiologicalrolesand pages 7-9). The periplasmic location is essential for its role in the ABC transport process: FliY captures extracellular cystine that has crossed the outer membrane, binds it with high affinity in the periplasmic space, and then delivers the substrate to the inner-membrane-spanning YecSC complex for ATP-driven translocation into the cytoplasm. This localization is consistent with the characteristic architecture of bacterial ABC importers, which universally employ periplasmic or extracellular substrate-binding proteins to achieve high substrate affinity and specificity.

Biological Pathways and Metabolic Context

Sulfur Assimilation Pathway

The FliY-YecSC transporter plays a central role in bacterial sulfur assimilation, particularly under conditions of sulfur limitation (zhou2020escherichiacolik12 pages 1-2, imlay2015physiologicalrolesand pages 1-2). The pathway proceeds as follows:

Cystine uptake: FliY-YecSC imports L-cystine from the extracellular environment
Reduction: Imported cystine is rapidly reduced to L-cysteine in the cytoplasm through glutathione-dependent processes (imlay2015physiologicalrolesand pages 4-5)
Central sulfur metabolism: The resulting cysteine pool serves as the hub for synthesis of all cellular sulfur-containing biomolecules, including methionine, iron-sulfur clusters, glutathione, lipoate, biotin, and other essential cofactors (zhou2020escherichiacolik12 pages 1-2, zhou2020escherichiacolik12 pages 2-4)

This pathway is particularly critical in aerobic environments where extracellular cysteine spontaneously oxidizes to cystine (zhou2020escherichiacolik12 pages 1-2). Under these conditions, cystine import via FliY-YecSC provides a more economical route to sulfur acquisition compared to sulfate assimilation, which requires eight electrons and substantial ATP expenditure to reduce sulfate to sulfide before incorporation into cysteine (zhou2020escherichiacolik12 pages 1-2, zhou2020escherichiacolik12 pages 2-4).

Transcriptional Regulation

Expression of the fliY gene is tightly regulated in response to cellular sulfur status through the CysB transcriptional regulator (imlay2015physiologicalrolesand pages 7-9). When bacteria grow on poor sulfur sources such as sulfate, fliY transcription is induced 3- to 10-fold compared to growth on cystine (imlay2015physiologicalrolesand pages 7-9). This induction is mediated by CysB, which becomes activated when intracellular cysteine pools decline and N-acetylserine accumulates (imlay2015physiologicalrolesand pages 1-2).

Notably, the regulatory architecture of the FliY-YecSC system exhibits an unusual feature: while fliY itself is strongly induced by CysB during sulfur limitation, the genes encoding its membrane partners (yecS and yecC) are predominantly transcribed from a separate, CysB-independent promoter upstream of the dcyD gene (imlay2015physiologicalrolesand pages 7-9). This differential regulation results in increased FliY protein levels during sulfur starvation while membrane transporter levels remain relatively constant. Transport kinetic studies demonstrated that under conditions of high cystine availability, the membrane complex (YecSC) rather than substrate binding becomes rate-limiting, explaining why increased FliY expression does not necessarily increase overall transport rates when cystine is abundant (imlay2015physiologicalrolesand pages 7-9).

Coordination with Alternative Transport Systems

Bacteria maintain two distinct cystine import systems with complementary properties. The FliY-YecSC (TcyJLN) ABC transporter handles nanomolar substrate concentrations with high affinity, while the YdjN (TcyP) proton-driven symporter operates effectively at micromolar concentrations (imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 9-10). Both systems are co-induced by CysB during sulfur limitation, providing bacteria with the capacity to efficiently scavenge cystine across a wide concentration range (imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 7-9). This dual-transporter strategy is phylogenetically widespread, occurring in Enterobacteriaceae and Firmicutes including Bacillus subtilis, suggesting strong evolutionary selection for this arrangement (imlay2015physiologicalrolesand pages 9-10).

Gene Nomenclature and Evolutionary Context

An important clarification regarding the fliY gene name: despite the "fli" prefix typically associated with flagellar genes, FliY has no functional relationship to flagellar assembly or motility (imlay2015physiologicalrolesand pages 5-7). The name derives solely from the gene's chromosomal proximity to authentic flagellar genes (fliA, fliZ) in E. coli and related bacteria. Functional studies clearly demonstrate that FliY's primary and likely exclusive role is in cystine transport for sulfur assimilation (imlay2015physiologicalrolesand pages 5-7, imlay2015physiologicalrolesand pages 7-9). In recognition of this, researchers have proposed renaming the system as TcyJLN (Transport Cystine J, L, N), where FliY corresponds to TcyJ (imlay2015physiologicalrolesand pages 1-2).

Structural and Biochemical Features

Protein Family Assignment

FliY belongs to the bacterial solute-binding protein family 3 (SBP_bac_3), as indicated by its Pfam domain PF00497 (UniProt annotation). This family is characterized by a conserved fold and substrate-binding mechanism shared among periplasmic binding proteins associated with ABC transporters. Members of this family typically employ a "Venus flytrap" conformational change mechanism, where substrate binding induces closure of two protein domains around the ligand, creating a high-affinity binding site.

Amino Acid Composition and Functional Constraints

A striking biochemical feature of cystine transport proteins is their exceptionally low cysteine content. Analysis of the E. coli FliY-YecSC system revealed that these four proteins collectively contain only one cysteine residue (0.08%) among 1,201 total amino acids, compared to 1.7% cysteine in the overall E. coli proteome (imlay2015physiologicalrolesand pages 9-10). This 20-fold underrepresentation of cysteine is extreme even compared to other amino acid transporters, which average 4.8% content of their cognate substrate amino acid (imlay2015physiologicalrolesand pages 9-10).

This compositional bias reflects an evolutionary adaptation to the protein's function in sulfur-starved cells: when cellular cysteine pools are depleted, proteins must be synthesizable despite limited cysteine availability (imlay2015physiologicalrolesand pages 9-10). The single exceptional cysteine residue in the E. coli YdjN transporter (at position 346) is functionally essential and cannot be replaced by serine or threonine without complete loss of activity, indicating it plays a critical catalytic or structural role (imlay2015physiologicalrolesand pages 9-10).

Evidence Basis and Inference for Xenorhabdus nematophila

While the functional characterization presented above derives primarily from studies in E. coli, several lines of evidence support extrapolation to Xenorhabdus nematophila:

High sequence conservation: The FliY-YecSC system is phylogenetically widespread across Enterobacteriaceae and shows conservation even in more distantly related Firmicutes (imlay2015physiologicalrolesand pages 9-10)
Conserved genomic context: The genomic organization of fliY and associated genes (yecS, yecC) is broadly conserved among bacteria (imlay2015physiologicalrolesand pages 5-7)
Consistent protein family assignment: The Xenorhabdus nematophila protein (D3VIU4) is correctly annotated as belonging to the same protein family (bacterial solute-binding protein 3) and possessing the same domains as characterized E. coli FliY
Functional requirement: As an entomopathogenic bacterium, Xenorhabdus nematophila faces sulfur acquisition challenges in diverse environments (insect hosts, nematode symbionts, soil), making high-affinity cystine transport systems functionally essential

Summary Table of Key Properties

Property	Description	Evidence/Citation
Protein Function	Periplasmic substrate-binding protein component of the FliY-YecSC ABC importer; captures extracellular cystine and delivers it to the membrane transporter for ATP-dependent uptake. Loss of both fliY and ydjN abolishes detectable cystine import and prevents growth on cystine as sole sulfur source.	(imlay2015physiologicalrolesand pages 4-5, imlay2015physiologicalrolesand pages 5-7)
Substrate Specificity	Primary physiological substrate is L-cystine (oxidized cysteine). FliY-YecSC can also transport diaminopimelate (DAP), indicating broader specificity for related diamino compounds, but cystine is the key sulfur-acquisition substrate.	(imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 9-10)
Binding Affinity (Km)	Apparent half-maximal transport for FliY-YecSC is ~30 nM cystine, about 100-fold lower than YdjN (~2 µM), making FliY-YecSC the high-affinity system used when cystine is scarce.	(imlay2015physiologicalrolesand pages 9-10)
Transport Partners	Functions with YecS and YecC as an ABC transport system: FliY is the periplasmic binding protein, YecS the ATPase-associated component, and YecC the membrane-spanning transport component. yecS mutation phenocopies loss of the FliY system.	(imlay2015physiologicalrolesand pages 5-7)
Cellular Localization	Soluble periplasmic binding protein in Gram-negative bacteria; operates in the periplasm to bind cystine before transfer to the inner-membrane YecSC complex.	(imlay2015physiologicalrolesand pages 5-7, imlay2015physiologicalrolesand pages 7-9)
Protein Family	Member of the bacterial solute-binding protein family associated with ABC importers; specifically the high-affinity cystine-binding component of the FliY-YecSC/TcyJLN system.	(imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 5-7)
Regulation	fliY transcription is induced ~3- to 10-fold in sulfate medium versus cystine medium and is controlled by the sulfur starvation regulator CysB. Constitutively active CysB upregulates fliY even in cystine medium.	(imlay2015physiologicalrolesand pages 7-9)
Biological Pathway	Participates in sulfur assimilation: cystine import under sulfur limitation, followed by intracellular reduction to cysteine, which feeds synthesis of methionine, glutathione, iron-sulfur clusters, and other sulfur-containing metabolites. It is especially important at nanomolar cystine concentrations.	(zhou2020escherichiacolik12 pages 1-2, imlay2015physiologicalrolesand pages 1-2, imlay2015physiologicalrolesand pages 9-10)
Alternative Names	Proposed renaming in E. coli: FliY-YecSC as TcyJLN, where “Tcy” denotes transport of cystine. The system is also described as the FliY-YecSC cystine ABC transporter.	(imlay2015physiologicalrolesand pages 1-2)
Gene Name Origin	The name fliY comes from its chromosomal position downstream of flagellar genes, but functional evidence shows it is not a flagellar structural/regulatory gene; its main role is cystine transport.	(imlay2015physiologicalrolesand pages 5-7)

Table: This table summarizes experimentally supported properties of FliY based mainly on E. coli and related bacterial evidence, highlighting its role as a high-affinity periplasmic cystine-binding protein. It is useful for inferring the likely function of the Xenorhabdus nematophila ortholog when direct species-specific studies are lacking.

Research Gaps and Future Directions

Despite the robust functional characterization in model organisms, several aspects specific to Xenorhabdus nematophila FliY warrant investigation:

Direct biochemical characterization of substrate specificity and binding affinity in the Xenorhabdus protein
Role of cystine/cysteine transport during the complex lifecycle transitions between nematode symbiosis and insect pathogenesis
Potential interactions with Xenorhabdus-specific regulatory networks beyond CysB
Contribution to virulence and host colonization, given the importance of sulfur metabolism in pathogenesis

Conclusion

The fliY gene (D3VIU4) from Xenorhabdus nematophila encodes a periplasmic substrate-binding protein that functions as the high-affinity cystine-capture component of the FliY-YecSC (TcyJLN) ABC transporter system. Based on extensive functional studies in E. coli and phylogenetic conservation, this protein operates in the periplasmic space to bind L-cystine with nanomolar affinity (Km ~30 nM) and deliver it to membrane-associated ATPase and permease components for ATP-driven import. The imported cystine is reduced to cysteine, which serves as the central precursor for all cellular sulfur-containing biomolecules. Expression is induced during sulfur limitation through the CysB transcriptional regulator. Despite its misleading name (derived from chromosomal proximity to flagellar genes), FliY has no role in flagellar assembly and functions exclusively in sulfur assimilation. The protein belongs to the bacterial solute-binding protein family 3 and exhibits characteristic features including extremely low cysteine content to permit synthesis during sulfur starvation. This transport system is essential for bacterial survival under conditions of limiting sulfur availability, particularly when cystine concentrations are in the nanomolar range.

References

(imlay2015physiologicalrolesand pages 1-2): Karin R. Chonoles Imlay, Sergey Korshunov, and James A. Imlay. Physiological roles and adverse effects of the two cystine importers of escherichia coli. Journal of Bacteriology, 197:3629-3644, Dec 2015. URL: https://doi.org/10.1128/jb.00277-15, doi:10.1128/jb.00277-15. This article has 110 citations and is from a peer-reviewed journal.
(imlay2015physiologicalrolesand pages 5-7): Karin R. Chonoles Imlay, Sergey Korshunov, and James A. Imlay. Physiological roles and adverse effects of the two cystine importers of escherichia coli. Journal of Bacteriology, 197:3629-3644, Dec 2015. URL: https://doi.org/10.1128/jb.00277-15, doi:10.1128/jb.00277-15. This article has 110 citations and is from a peer-reviewed journal.
(zhou2020escherichiacolik12 pages 1-2): Yidan Zhou and James A. Imlay. Escherichia coli k-12 lacks a high-affinity assimilatory cysteine importer. mBio, Jun 2020. URL: https://doi.org/10.1128/mbio.01073-20, doi:10.1128/mbio.01073-20. This article has 27 citations and is from a domain leading peer-reviewed journal.
(imlay2015physiologicalrolesand pages 4-5): Karin R. Chonoles Imlay, Sergey Korshunov, and James A. Imlay. Physiological roles and adverse effects of the two cystine importers of escherichia coli. Journal of Bacteriology, 197:3629-3644, Dec 2015. URL: https://doi.org/10.1128/jb.00277-15, doi:10.1128/jb.00277-15. This article has 110 citations and is from a peer-reviewed journal.
(imlay2015physiologicalrolesand pages 7-9): Karin R. Chonoles Imlay, Sergey Korshunov, and James A. Imlay. Physiological roles and adverse effects of the two cystine importers of escherichia coli. Journal of Bacteriology, 197:3629-3644, Dec 2015. URL: https://doi.org/10.1128/jb.00277-15, doi:10.1128/jb.00277-15. This article has 110 citations and is from a peer-reviewed journal.
(imlay2015physiologicalrolesand pages 9-10): Karin R. Chonoles Imlay, Sergey Korshunov, and James A. Imlay. Physiological roles and adverse effects of the two cystine importers of escherichia coli. Journal of Bacteriology, 197:3629-3644, Dec 2015. URL: https://doi.org/10.1128/jb.00277-15, doi:10.1128/jb.00277-15. This article has 110 citations and is from a peer-reviewed journal.
(zhou2020escherichiacolik12 pages 2-4): Yidan Zhou and James A. Imlay. Escherichia coli k-12 lacks a high-affinity assimilatory cysteine importer. mBio, Jun 2020. URL: https://doi.org/10.1128/mbio.01073-20, doi:10.1128/mbio.01073-20. This article has 27 citations and is from a domain leading peer-reviewed journal.

Artifacts

Edison artifact artifact-00

Citations

imlay2015physiologicalrolesand pages 5-7
imlay2015physiologicalrolesand pages 9-10
imlay2015physiologicalrolesand pages 4-5
imlay2015physiologicalrolesand pages 7-9
imlay2015physiologicalrolesand pages 1-2
https://doi.org/10.1128/jb.00277-15,
https://doi.org/10.1128/mbio.01073-20,

OpenScientist

(D3VIU4-hypotheses/prediction-ligand-gated-ion-channel/openscientist.md)

AIGR Gene Hypothesis Deep Research — Final Report OpenScientist openscientist-autonomous 1 citations 3 artifacts 2026-07-09T06:48:41.924451 citations file

OpenScientist final report (text/html)

OpenScientist final report (application/pdf)

OpenScientist d3viu4 evidence matrix (text/tab-separated-values)

AIGR Gene Hypothesis Deep Research — Final Report

Gene: D3VIU4 (gene symbol fliY, locus XNC1_0570)
Organism: Xenorhabdus nematophila ATCC 19061 (NCBITaxon:406817)
UniProt: D3VIU4
Hypothesis under evaluation: ProtNLM2 predicts ligand-gated monoatomic ion channel activity (GO:0015276)
Focus type: computational_prediction

Summary

The ProtNLM2 machine-learning prediction that D3VIU4 possesses ligand-gated monoatomic ion channel activity (GO:0015276) is REFUTED. Five independent, orthogonal lines of evidence — domain architecture, curated localization, sequence hydropathy, cross-species orthology, and predicted 3D structure — converge on a single, coherent identity: D3VIU4 is a soluble periplasmic solute-binding protein (SBP) of ABC-transporter family 3, specifically a FliY/TcyA-type L-cystine-binding protein. It is the extracytoplasmic substrate-recognition subunit of an ABC importer: it captures cystine/cysteine in the periplasm and delivers it to a separate membrane permease. It is not a channel, does not span the membrane, and has no ion-conducting pore.

The most compelling reason for confidence is convergence. UniProt annotates the protein as a periplasmic "Cysteine transport protein (ABC superfamily, peri_bind)" with a cleaved signal peptide and a single SBP domain; a computed Kyte–Doolittle hydropathy profile finds zero transmembrane segments; the protein is 54.6% identical over its full length to the experimentally characterized E. coli FliY cystine-binding protein; and its AlphaFold model is a compact globular single domain with no membrane-spanning helix. A ligand-gated ion channel requires several pore-lining transmembrane helices and an oligomeric ion-conducting pore. D3VIU4 has none of these features.

The prediction is best explained as a fold-homology misassignment. The periplasmic binding-protein type II fold (SUPFAM SSF53850) shared by SBP family 3 proteins is evolutionarily homologous to the extracellular ligand-binding domain (LBD) of ionotropic glutamate receptors — a genuine ligand-gated ion channel family. A predictor that keys on the ligand-binding clamshell without confirming the presence of the (absent) transmembrane channel module can spuriously transfer the channel label. The single caveat is that structural evidence is computational (AlphaFold) plus homology transfer from E. coli; there is no direct biochemical assay on the X. nematophila protein itself. This does not change the verdict, because the complete absence of any transmembrane segment is a hard architectural disqualifier for channel activity.

Key Findings

Finding 1 — D3VIU4 is a periplasmic SBP family 3 (FliY/TcyA cystine-binding) protein, not an ion channel

The UniProt record for D3VIU4 assigns the gene name fliY (locus XNC1_0570) and the submission name "Cysteine transport protein (ABC superfamily, peri_bind)". Every domain-level annotation available for this protein points to a single, coherent identity as a bacterial extracellular solute-binding protein:

Resource	Signature	Meaning
Pfam	PF00497 (SBP_bac_3)	Bacterial extracellular solute-binding protein, family 3
InterPro	IPR001638 (Solute-binding_3/MltF_N) + IPR018313	SBP family 3 domain and conserved signature
CDD	cd13711 (PBP2_Ngo0372_TcyA)	Type 2 periplasmic binding fold, cystine-binding subfamily
SUPFAM	SSF53850	Periplasmic binding protein-like II
Gene3D	3.40.190.10	Periplasmic binding protein-like fold

Critically, the UniProt feature table describes exactly the architecture of a secreted binding protein and nothing resembling a channel: a cleaved N-terminal signal peptide (residues 1–28), a mature chain (residues 29–262), and a single SBP domain (residues 40–259). The curated subcellular location is outer membrane-bounded periplasmic space (GO:0030288) — soluble, on the outside of the inner membrane, exactly where an ABC-importer binding protein operates.

A computed Kyte–Doolittle hydropathy profile (window 19, transmembrane threshold 1.6) found zero transmembrane segments. The only hydrophobic peak (residues 11–18, maximum value 2.32 at residue 17) lies entirely within the cleaved signal peptide (1–28); it is a secretion signal, not a membrane anchor. There are no internal transmembrane helices and therefore no pore-forming architecture. A ligand-gated ion channel requires, at minimum, several membrane-spanning helices that line an ion-conducting pore. D3VIU4 has none.

Finding 2 — D3VIU4 is a full-length ortholog (54.6% identity) of the experimentally characterized E. coli FliY cystine-binding protein

To transfer function with confidence, orthology must be established across the whole protein, not a local patch. A Needleman–Wunsch global alignment of D3VIU4 (262 aa) against E. coli K-12 FliY (UniProt P0AEM9, 266 aa) yields 131 identical residues over 240 aligned columns = 54.6% identity, distributed across the entire length of the protein. This is unambiguous full-length orthology, well above the ~30% threshold typically used for confident functional transfer of a single-domain protein.

E. coli FliY is not a hypothetical protein — it is the experimentally characterized periplasmic L-cystine-binding protein of the FliY–YecSC ABC importer. In that system, the soluble FliY captures L-cystine in the periplasm with high affinity (K_m = 110 nM), and the membrane permease YecS (with the ATPase YecC) transports it across the inner membrane. Because D3VIU4 is 54.6% identical to FliY over its full length, the following properties transfer directly: soluble periplasmic localization, a cleaved signal peptide, a single SBP_bac_3 fold, cystine/cysteine ligand binding, and a substrate-delivery role within an ABC importer. None of these is a channel property.

A minor technical note: a hand-transcribed PROSITE PS01039 regex did not match the sequence, but PROSITE pattern transcription by hand is error-prone, and the pattern match is not required for the identification given the concordant InterPro/CDD/Pfam calls and the 54.6% full-length ortholog identity. This does not weaken the conclusion.

Finding 3 — The AlphaFold model is a single compact globular SBP domain with no transmembrane helix

Structure provides the most direct test of "channel vs. soluble binding protein," because channels are elongated, membrane-embedded, and pore-bearing, whereas SBPs are compact and globular. The AlphaFold DB model AF-D3VIU4-F1 (v6), 262 residues, has an overall mean pLDDT of 90.7 (high confidence).

Analysis of the model geometry:

Mature core (residues 29–262): a single compact globular domain, radius of gyration 17.8 Å, bounding box 41.5 × 35.2 × 48.5 Å, anisotropy (max/median dimension) = 1.17 — near-spherical/globular, exactly the shape of a bilobed SBP. Mean pLDDT 96.0 (very high confidence).
Signal peptide (residues 1–28): mean pLDDT 46.5 (low, flexible/disordered), with its centroid sitting 58.8 Å from the folded-core centroid — a protruding, disordered appendage (the uncleaved signal in the model), not part of any structured pore.
Membrane-spanning-helix test: the single most hydrophobic 19-residue window over the entire sequence has a mean Kyte–Doolittle value of 1.58, below the 1.6 TM threshold. No hydrophobic membrane-spanning helix exists anywhere in the protein.

The overall geometry is that of a bilobed soluble periplasmic binding protein (the SBP "Venus flytrap" clamshell), not an elongated multi-pass transmembrane channel. This independently and structurally confirms the sequence- and homology-based conclusions.

Mechanistic Model / Interpretation

The three findings converge on a single, well-supported model. D3VIU4 is the soluble substrate-binding subunit of a cystine ABC importer. It works in the periplasm, upstream of and physically separate from the membrane transport machinery:

   PERIPLASM (outside inner membrane)
   ┌─────────────────────────────────────────────┐
   │      L-cystine                              │
   │          \    ┌───────────────┐             │
   │           └──►│   D3VIU4       │  soluble    │
   │               │  (FliY/TcyA)   │  binding    │
   │               │  SBP family 3  │  protein    │
   │               └──────┬────────┘  (NO pore)   │
   │                      │ delivers cystine      │
   ══════════════════════▼══════════════════════   INNER MEMBRANE
┌──────────────────────────────┐
│   YecS permease (TM pore)     │  <-- the actual
└───────────────┬──────────────┘      membrane transporter
┌───────────────▼──────────────┐
│   YecC ATPase (cytoplasm)     │
└───────────────────────────────┘
   CYTOPLASM

The key architectural distinctions for curation:

Feature	Ligand-gated ion channel (GO:0015276)	D3VIU4 (observed)
Transmembrane helices	Multiple pore-lining TM helices	Zero (only a cleaved signal peptide)
Oligomeric transmembrane pore	Yes	No
Ion selectivity filter	Yes	No
Localization	Integral membrane	Soluble periplasmic (GO:0030288)
Fold	Ion-channel/LBD assembly	Compact globular SBP type II (SSF53850)
Ligand role	Ligand gates ion flux	L-cystine bound & delivered — no flux gated

Why the misassignment happened. The periplasmic binding-protein type II fold (SSF53850) that defines SBP family 3 is evolutionarily homologous to the extracellular ligand-binding domain (LBD) of ionotropic glutamate receptors, which are ligand-gated ion channels. The two share a bilobed "Venus flytrap" clamshell that closes around an amino-acid ligand. A predictor that recognizes the ligand-binding clamshell but does not verify the presence of the separate transmembrane pore module can therefore over-transfer the ion-channel label. D3VIU4 has the binding clamshell but none of the channel/pore module — so the transfer is incorrect. This is a textbook example of the "shared ligand-binding module without the channel module" failure mode that the research objective explicitly asks to consider.

Evidence Matrix

#	Citation	Evidence type	Supports/Refutes	Claim tested	Key finding	Context	Confidence & limitations
1	UniProt D3VIU4 record	Review/database	Refutes channel; supports SBP	Domain architecture	Gene fliY; "Cysteine transport protein (ABC superfamily, peri_bind)"; Pfam PF00497, InterPro IPR001638, CDD cd13711 (TcyA cystine-binding), SUPFAM SSF53850; signal peptide 1–28, mature 29–262, single SBP domain; location GO:0030288	X. nematophila annotation	High for architecture; database-level, not a direct assay
2	Computed Kyte–Doolittle hydropathy (this work)	Computational	Refutes channel	TM pore helices present?	Zero TM segments; only hydrophobic peak inside cleaved signal peptide (1–28)	Full 262-aa sequence	High; standard method, TM threshold 1.6
3	PMID: 25837721	Direct assay (ortholog)	Refutes channel; supports cystine-binding SBP	Function of FliY ortholog	FliY is the periplasmic L-cystine-binding protein of ABC importer YecSC (K_m = 110 nM)	E. coli	High for E. coli; transferred by 54.6% identity
4	Needleman–Wunsch alignment vs E. coli FliY / P0AEM9 (this work)	Structural/evolutionary	Refutes channel; supports SBP	Orthology / functional transfer	54.6% identity (131/240) full-length to characterized FliY	XENNA vs ECOLI	High; full-length, above transfer threshold
5	AlphaFold DB AF-D3VIU4-F1 v6 + geometry (this work)	Structural/computational	Refutes channel; supports SBP	Globular vs membrane pore	Compact globular single domain, Rg 17.8 Å, anisotropy 1.17, core pLDDT 96; no membrane-spanning helix (max window 1.58 < 1.6)	Predicted structure	High-confidence model (mean pLDDT 90.7); computational, not experimental
6	ProtNLM2 (seed)	Computational	Competing (refuted)	Ligand-gated ion channel activity	Predicts GO:0015276 — contradicted by all above	Automated prediction	Low; fold-homology artifact

Evidence Base (Literature)

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli. PMID: 25837721. This paper directly characterizes FliY — the full-length ortholog of D3VIU4 (54.6% identity) — as "the periplasmic L-cystine-binding protein" delivering L-cystine (K_m = 110 nM) to the membrane ABC transporter YecSC. Verified snippet: "the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein." This anchors the functional identity of D3VIU4 as a soluble binding protein and directly contradicts the ion-channel prediction, because a periplasmic binding protein is architecturally and functionally distinct from a ligand-gated channel.

Database/orientation resources used: UniProt (D3VIU4, P0AEM9), Pfam (PF00497), InterPro (IPR001638/IPR018313), CDD (cd13711 TcyA), SUPFAM (SSF53850), Gene3D (3.40.190.10), and AlphaFold DB (AF-D3VIU4-F1 v6). These are treated as orientation/database-level support; the decisive experimental anchor is the E. coli FliY characterization above, transferred via full-length orthology.

GO Curation Implications

Lead (requires curator verification):

GO:0015276 (ligand-gated monoatomic ion channel activity) — REMOVE / do not propagate. The ProtNLM2 prediction is a fold-homology misassignment. There is no transmembrane pore, no channel architecture, and no supporting experimental or structural evidence. Retaining it would introduce a false molecular-function claim.

Recommended replacements, supported by the evidence:

Aspect	Recommended GO term	Evidence basis
MF	Amino-acid binding / L-cystine binding (e.g., GO:0140328 amino acid binding, or cystine binding)	Orthology to FliY cystine-binding protein (PMID:25837721); CDD TcyA cystine-binding subfamily
BP	L-cystine / amino-acid import across plasma membrane (GO:0015811 L-cystine transport; GO:0006865 amino acid transport)	FliY–YecSC importer role; fliY gene identity
CC	Periplasmic space (GO:0030288); part of ABC transporter complex (GO:0043190)	UniProt-curated location; soluble SBP subunit of ABC importer

The MF should reflect ligand binding and substrate delivery, not transport activity in isolation (D3VIU4 is not the permease). Avoid the uninformative "protein binding"; a specific amino-acid/cystine-binding term is supported.

Mechanistic Scope

The molecular function directly tested is ion-channel (pore-forming, gated ion-conducting) activity versus soluble ligand binding. The evidence localizes D3VIU4's immediate activity to binding L-cystine in the periplasm and delivering it to a membrane permease — a recognition/capture function. It is not itself a transporter that moves ions across a membrane, and it is not a channel that gates ion flux in response to a ligand. Any "ion/solute transport" is a property of the whole multi-subunit ABC complex driven by ATP hydrolysis at the ABC ATPase — mechanistically distinct from gated ion-channel conductance and not attributable to D3VIU4 alone.

Downstream/indirect roles that should not be conflated with the direct MF: the E. coli ortholog's contribution to oxidative-stress defense (via cystine uptake feeding cytoplasmic cysteine/glutathione pools) is a pathway consequence of the importer, not a molecular activity of the binding protein. Any organismal phenotype in X. nematophila (e.g., nematode symbiosis or insect pathogenesis) would likewise be a downstream, context-specific outcome, not evidence for channel activity.

Conflicts and Alternatives

The only "conflict" is the ProtNLM2 prediction itself, which is contradicted by all five orthogonal analyses. No primary literature, database annotation, or structural feature supports channel activity.
Fold homology as the source of the error: SBP family 3 shares the SSF53850 clamshell fold with ionotropic glutamate receptor LBDs (genuine ligand-gated ion channels). This is the most plausible mechanistic explanation for the spurious label — the predictor recognized the ligand-binding module but not the (absent) channel module. This is fold-family over-annotation, not a real functional signal.
No isoform or paralog confusion within X. nematophila is needed to explain the finding: the single UniProt record and single AlphaFold model both give the same SBP answer.
Cross-species transfer caveat: the direct biochemical characterization (PMID:25837721) is in E. coli, not X. nematophila. However, 54.6% full-length identity is well within confident-transfer range, and the architectural disqualifier (no TM segment) is intrinsic to the X. nematophila sequence itself.

Limitations and Knowledge Gaps

No direct biochemical assay on the X. nematophila protein. Cystine-binding affinity, ligand specificity, and importer partnership are inferred from the E. coli ortholog and domain calls, not measured for D3VIU4. What was checked: orthology (54.6%), domain signatures, AlphaFold geometry. Why it matters: confirms substrate identity (cystine vs another amino acid). Resolution: isothermal titration calorimetry or fluorescence-based ligand binding on purified recombinant D3VIU4.
Structural evidence is computational (AlphaFold), not experimental. Why it matters: although pLDDT is high (90.7 overall, 96 core), an experimental structure would remove residual model uncertainty. Resolution: X-ray/cryo-EM of D3VIU4, ideally with bound cystine.
The cognate permease/ATPase in X. nematophila were not explicitly mapped. Why it matters: confirms the ABC-importer context (YecS/YecC orthologs) and the BP/CC assignment. Resolution: genomic neighborhood analysis of XNC1_0570 and operon reconstruction.
Exact substrate could be cystine, cysteine, or a related amino acid. The CDD TcyA subfamily and FliY orthology point to cystine, but exhaustive binding-pocket residue analysis was not performed. This does not affect the channel refutation.

None of these gaps affects the central verdict: the absence of any transmembrane pore-forming segment is decisive against ion-channel activity.

Discriminating Tests

TMHMM/DeepTMHMM on the mature sequence — expect 0 TM segments, consistent with the computed KD result.
AlphaFold-DB model inspection — expect a bilobed SBP fold with no TM helix bundle (confirmed here).
Genome-neighborhood analysis for adjacent ABC permease (yecS) and ATPase (yecC) genes in X. nematophila — confirms the ABC-importer context.
Ligand-binding assay (ITC/fluorescence) with L-cystine on purified D3VIU4 — positive binding (expected K_d nM–µM by analogy to FliY's K_m = 110 nM) confirms SBP function.
Electrophysiology (single-channel recording / liposome reconstitution) — the discriminating negative test: a true ligand-gated ion channel would produce gated ionic currents; D3VIU4 should produce none, directly refuting GO:0015276.
Complementation of an E. coli ΔfliY strain with D3VIU4 — functional rescue would confirm conserved cystine-binding-protein function.

Proposed Follow-up Actions / Curation Leads

(All labeled as leads requiring curator verification.)

Action change: Reject/withdraw the ProtNLM2 annotation GO:0015276 for D3VIU4 as a computational misassignment (fold homology to iGluR ligand-binding domains, without the transmembrane channel module).
Candidate replacement MF term: amino-acid / L-cystine binding (transporter-substrate-binding activity), based on FliY orthology and CDD TcyA subfamily.
Candidate BP term: L-cystine / amino-acid import across the plasma membrane as the periplasmic SBP subunit of an ABC importer.
Candidate CC term: periplasmic space (GO:0030288); part of ABC transporter complex (GO:0043190).
Candidate reference to verify: PMID: 25837721 — snippet: "the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein."
Suggested curator questions: (1) Does the review framework prefer a specific "cystine binding" MF term or the general "amino-acid binding"? (2) Should the annotation carry a "part_of ABC transporter complex" contextual qualifier?
Suggested experiments: ITC cystine-binding assay; liposome electrophysiology (expected null result); ΔfliY complementation in E. coli.

Bottom Line

The ProtNLM2 prediction of ligand-gated monoatomic ion channel activity (GO:0015276) for D3VIU4 is REFUTED. D3VIU4 is a soluble periplasmic L-cystine-binding protein (SBP family 3, FliY/TcyA type) — the substrate-recognition subunit of an ABC importer — with a cleaved signal peptide, zero transmembrane segments, a compact globular AlphaFold fold, and 54.6% full-length identity to the experimentally characterized E. coli FliY. It lacks the transmembrane pore-forming architecture required for any ion channel. Curators should remove GO:0015276 and annotate amino-acid/cystine binding (MF), cystine/amino-acid import (BP), and periplasmic space (CC) instead.

Artifacts

📄 View Raw YAML

id: D3VIU4
gene_symbol: D3VIU4
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:406817
  label: Xenorhabdus nematophila (strain ATCC 19061 / DSM 3370 / CCUG 14189 / LMG
    1036 / NCIMB 9965 / AN6)
description: >-
  FliY (locus tag XNC1_0570) is a periplasmic solute-binding protein of the bacterial
  solute-binding protein family 3 (SBP_bac_3, PF00497) in the Gram-negative entomopathogenic
  bacterium Xenorhabdus nematophila. It functions as the substrate-capture component of
  the FliY-YecSC (TcyJLN) ATP-binding cassette (ABC) transporter system, which imports
  L-cystine with high affinity (Km ~30 nM in E. coli orthologs). FliY is synthesized with
  an N-terminal signal peptide (residues 1-28) that directs its secretion into the periplasmic
  space, where it binds extracellular L-cystine and delivers it to the inner-membrane-associated
  YecSC complex for ATP-driven translocation into the cytoplasm. The imported cystine is
  reduced to L-cysteine, which serves as a central hub for synthesis of methionine, glutathione,
  iron-sulfur clusters, and other sulfur-containing biomolecules. Despite the "fliY" gene name
  (which derives solely from chromosomal proximity to flagellar genes), this protein has no
  role in flagellar assembly or motility; its function is restricted to cystine transport
  for sulfur assimilation. The protein is 262 amino acids long and contains the conserved
  Solute-binding protein family 3/N-terminal domain (IPR001638).
existing_annotations:
- term:
    id: GO:0030288
    label: outer membrane-bounded periplasmic space
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  qualifier: located_in
  review:
    summary: >-
      FliY is a periplasmic solute-binding protein in Gram-negative bacteria. It contains
      an N-terminal signal peptide (residues 1-28) directing export across the inner membrane
      into the periplasm. As a component of the FliY-YecSC ABC transporter, it operates as
      a soluble protein in the outer membrane-bounded periplasmic space, where it captures
      L-cystine and delivers it to the inner-membrane YecSC complex. This localization is
      well-supported by the protein's signal peptide, its membership in the bacterial
      solute-binding protein 3 family, and the conserved architecture of Gram-negative
      ABC import systems. The ARBA prediction is consistent with the known biology.
    action: ACCEPT
- term:
    id: GO:0030313
    label: cell envelope
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: >-
      The cell envelope annotation is derived from UniProtKB-SubCell mapping and is not
      incorrect -- FliY does reside within the cell envelope. However, this is a very broad
      term (GO:0030313) that encompasses the outer membrane, periplasmic space, inner membrane,
      and cell wall. The more specific annotation to outer membrane-bounded periplasmic space
      (GO:0030288) already captures the precise localization of FliY. Retaining this annotation
      as non-core since it is accurate but less informative than the periplasmic space annotation.
    action: KEEP_AS_NON_CORE
- term:
    id: GO:0016597
    label: amino acid binding
  evidence_type: ISS
  original_reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
  qualifier: enables
  review:
    summary: >-
      FliY is the periplasmic substrate-binding protein of the FliY-YecSC (TcyJLN) ABC
      transporter. Its primary molecular function is binding L-cystine with high affinity
      in the periplasm. The amino acid binding term captures this substrate-binding role.
      This is inferred from sequence similarity to the well-characterized E. coli FliY
      ortholog and from membership in the bacterial solute-binding protein family 3. The
      CDD annotation (cd13711, PBP2_Ngo0372_TcyA) further supports this functional assignment.
    action: NEW
    supported_by:
    - reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
      supporting_text: >-
        [Inferred from E. coli ortholog] FliY serves as the periplasmic substrate-binding
        component of an ATP-binding cassette (ABC) transporter complex designated FliY-YecSC.
        This system functions as a high-affinity importer of L-cystine.
- term:
    id: GO:0015811
    label: L-cystine transport
  evidence_type: ISS
  original_reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
  qualifier: involved_in
  review:
    summary: >-
      FliY functions as the substrate-capture component of the cystine ABC transporter
      system. Its role in L-cystine transport is inferred from sequence similarity to
      the E. coli FliY ortholog, which has been experimentally demonstrated to be essential
      for cystine import via the FliY-YecSC system. The UniProt submission name ("Cysteine
      transport protein") and CDD domain hit (PBP2_Ngo0372_TcyA) are consistent with this
      functional assignment.
    action: NEW
    supported_by:
    - reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
      supporting_text: >-
        [Inferred from E. coli ortholog] Genetic deletion studies definitively established
        that FliY, together with a second cystine transporter YdjN (TcyP), constitutes one
        of only two cystine import systems in the bacterium. Double mutants lacking both
        fliY and ydjN are completely unable to import detectable levels of cystine.
- term:
    id: GO:0043190
    label: ATP-binding cassette (ABC) transporter complex
  evidence_type: ISS
  original_reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
  qualifier: part_of
  review:
    summary: >-
      FliY is the periplasmic binding protein subunit of the FliY-YecSC ABC transporter
      complex. This complex membership is inferred from homology to the E. coli system
      where FliY, YecS, and YecC form the three-component ABC transporter, and from the
      UniProt annotation as an ABC superfamily periplasmic binding protein.
    action: NEW
    supported_by:
    - reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
      supporting_text: >-
        [Inferred from E. coli ortholog] The FliY-YecSC system operates through the canonical
        ABC transporter mechanism involving three protein components: FliY is the periplasmic
        substrate-binding protein, YecS the ATPase component, and YecC the integral membrane
        permease.
references:
- 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: PMID:22125637
  title: "The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus:\
    \ convergent lifestyles from divergent genomes"
  findings:
  - statement: >-
      This is the genome sequencing paper for Xenorhabdus nematophila strain ATCC 19061,
      which identified the fliY gene (locus tag XNC1_0570) encoding the cysteine transport
      protein.
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Genome paper that is the source of the D3VIU4 protein sequence. Does not contain
      functional characterization of FliY specifically, but provides the genomic context.
- id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
  title: "Deep research report on D3VIU4 fliY from Xenorhabdus nematophila"
  publication_type: DEEP_RESEARCH
  findings:
  - statement: >-
      FliY serves as the periplasmic substrate-binding component of the FliY-YecSC
      (TcyJLN) ABC transporter complex for high-affinity L-cystine import. Studies
      in E. coli demonstrate that the FliY-YecSC system achieves half-maximal transport
      at approximately 30 nM cystine, about 100-fold higher affinity than the alternative
      YdjN transporter. The protein belongs to bacterial solute-binding protein family 3
      and operates in the periplasmic space of Gram-negative bacteria.
core_functions:
- description: >-
    FliY is the periplasmic substrate-binding protein component of the FliY-YecSC
    (TcyJLN) ABC transporter complex. It captures L-cystine in the periplasm with
    high affinity and delivers it to the membrane-associated transporter components
    for ATP-dependent import. This supports sulfur assimilation under conditions of
    sulfur limitation.
  supported_by:
  - reference_id: "file:XENNA/D3VIU4/D3VIU4-deep-research-falcon.md"
    supporting_text: >-
      [Inferred from E. coli ortholog characterization] FliY serves as the periplasmic
      substrate-binding component of an ATP-binding cassette (ABC) transporter complex
      designated FliY-YecSC (also known as TcyJLN). This system functions as a high-affinity
      importer of L-cystine. The FliY-YecSC system achieves half-maximal transport at
      approximately 30 nanomolar cystine concentration.
  molecular_function:
    id: GO:0016597
    label: amino acid binding
  contributes_to_molecular_function:
    id: GO:0015184
    label: L-cystine transmembrane transporter activity
  directly_involved_in:
  - id: GO:0015811
    label: L-cystine transport
  locations:
  - id: GO:0030288
    label: outer membrane-bounded periplasmic space
  in_complex:
    id: GO:0043190
    label: ATP-binding cassette (ABC) transporter complex

D3VIU4

Gene Description

Existing Annotations Review

Core Functions

References

Deep Research

Falcon

Comprehensive Research Report: Gene fliY (D3VIU4) from Xenorhabdus nematophila

Executive Summary

Protein Function and Molecular Mechanism

Primary Function: High-Affinity Cystine Transport

Transport Mechanism and Molecular Partners

Substrate Specificity and Binding Affinity

Cellular Localization

Biological Pathways and Metabolic Context

Sulfur Assimilation Pathway

Transcriptional Regulation

Coordination with Alternative Transport Systems

Gene Nomenclature and Evolutionary Context

Structural and Biochemical Features

Protein Family Assignment

Amino Acid Composition and Functional Constraints

Evidence Basis and Inference for Xenorhabdus nematophila

Summary Table of Key Properties

Research Gaps and Future Directions

Conclusion

Artifacts

Citations

OpenScientist

AIGR Gene Hypothesis Deep Research — Final Report

Summary

Key Findings

Finding 1 — D3VIU4 is a periplasmic SBP family 3 (FliY/TcyA cystine-binding) protein, not an ion channel

Finding 2 — D3VIU4 is a full-length ortholog (54.6% identity) of the experimentally characterized E. coli FliY cystine-binding protein

Finding 3 — The AlphaFold model is a single compact globular SBP domain with no transmembrane helix

Mechanistic Model / Interpretation

Evidence Matrix

Evidence Base (Literature)

GO Curation Implications

Mechanistic Scope

Conflicts and Alternatives

Limitations and Knowledge Gaps

Discriminating Tests

Proposed Follow-up Actions / Curation Leads

Bottom Line

Artifacts

📄 View Raw YAML