D3VIU4 (locus XNC1_0570, submitter gene name fliY) is a 262-residue periplasmic solute-binding protein of bacterial solute-binding protein family 3 (SBP_bac_3, PF00497) from the Gram-negative entomopathogenic bacterium Xenorhabdus nematophila. It has an N-terminal signal peptide (residues 1-28) and a single type II periplasmic-binding-protein fold (residues 40-259), and is predicted to act in the periplasm as the substrate-capture subunit of an ABC importer for L-cystine. The gene lies immediately upstream of an ABC ATPase (XNC1_0571, yecC) and a permease (XNC1_0572, yecS/TcyL-type), the same binding protein-ATPase-permease arrangement as the cystine uptake operons of other bacteria, and a second, related binding protein (XNC1_0574) lies just downstream. Within the PANTHER family it groups with Bacillus subtilis TcyA, the binding protein of the TcyABC L-cystine uptake system, rather than with the Escherichia coli high-affinity cystine-binding protein TcyJ/FliY, which belongs to a sister subfamily. It conserves the tyrosine and tryptophan binding-pocket residues of the structurally characterised L-cystine receptor Ngo0372 of Neisseria gonorrhoeae. Cystine binding by D3VIU4 itself, its affinity, and its exact ligand range (cystine versus related sulfur amino acids) have not been tested. Despite the fliY name, it has no known role in flagellar assembly or motility.
Existing Annotations Review
GO Term
Evidence
Action
Reason
GO:0030288 outer membrane-bounded periplasmic space
IEA GO_REF:0000117
ACCEPT
Summary: D3VIU4 has a predicted N-terminal signal peptide (SignalP, residues 1-28) and a single family-3 solute-binding domain, the architecture of soluble periplasmic binding proteins of Gram-negative ABC importers. The periplasmic location is therefore well supported by sequence features and family membership, independently of the ARBA rule that generated this row. PAINT also places GO:0030288 as an IBD at the PTHR35936 node PTN000438641, although D3VIU4 itself carries no IBA row.
Reason: The term is as specific as the evidence allows for a soluble SBP in a diderm bacterium and represents where the core binding function is executed.
Supporting Evidence:
file:XENNA/D3VIU4/D3VIU4-uniprot.txt
FT SIGNAL 1..28
file:XENNA/D3VIU4/D3VIU4-uniprot.txt
Belongs to the bacterial solute-binding protein 3 family.
Summary: Cell envelope is true for a periplasmic binding protein but is a general term covering the outer membrane, periplasm, cell wall and inner membrane. The evidence (signal peptide, soluble SBP architecture) supports the more specific periplasmic space term, which the gene already carries.
Reason: Correct but less specific than the evidence supports; refine to outer membrane-bounded periplasmic space (GO:0030288, a part_of descendant already asserted on this gene) rather than keeping the general term as non-core.
ISS file:XENNA/D3VIU4/D3VIU4-bioinformatics/RESULTS-rereview.md
NEW
Summary: D3VIU4 is in PANTHER PTHR35936:SF34 with B. subtilis TcyA, the binding protein of an L-cystine uptake ABC system, and conserves the two binding-pocket residues (Tyr49 and Trp87, equivalent to Ngo0372 Tyr59/Trp97) that line the L-cystine site of the N. gonorrhoeae receptor Ngo0372 (Kd 21 nM). PAINT places GO:0016597 as an IBD at the PTHR35936 node PTN000438641. GO has no L-cystine binding term, so amino acid binding is the most specific available MF term. This does not rely on the E. coli FliY/TcyJ ortholog claim of the earlier draft: E. coli TcyJ is in subfamily SF35, not SF34.
Reason: Ligand-binding function of a family-3 SBP, inferred from subfamily membership with TcyA, conserved pocket residues and conserved operon context; no direct binding assay exists for D3VIU4.
Ngo0372 Tyr58 and Trp96 (UniProt SV2 numbering; Tyr59/Trp97 in PMID:22138345, where Tyr59 is crucial for L-cystine binding) align to D3VIU4 Tyr49 and Trp87.
ISS file:XENNA/D3VIU4/D3VIU4-bioinformatics/RESULTS-rereview.md
NEW
Summary: As the substrate-binding subunit of an ABC importer, the SBP does part of the work of transport (capturing substrate and delivering it to the permease), so process participation is appropriate if the substrate assignment holds. The cystine assignment rests on subfamily membership with B. subtilis TcyA (TcyABC mediates L-cystine uptake), conserved Ngo0372 pocket residues, and an adjacent ATPase/permease pair matching the E. coli tcyJ-tcyN-tcyL arrangement. The substrate range has not been tested, and the E. coli work cautions that similarity to FliY does not guarantee cystine use.
Reason: Reasonable homology- and synteny-based inference with an explicit caveat on ligand specificity; kept, but no longer grounded on direct E. coli FliY orthology or its kinetics.
ISS file:XENNA/D3VIU4/D3VIU4-bioinformatics/RESULTS-rereview.md
NEW
Summary: D3VIU4 is the predicted substrate-binding subunit of the ABC importer encoded with the adjacent ATPase (XNC1_0571) and permease (XNC1_0572). The earlier draft proposed the parent GO:0043190 ABC transporter complex; the specific child for complexes containing a substrate-binding subunit is the more informative choice for an SBP.
Reason: Refined from the previously proposed GO:0043190 to its descendant GO:0055052, which describes exactly this kind of complex (SBP free in the periplasm, joining the membrane subunits when loaded).
XNC1_0570 (D3VIU4, SBP) is followed by XNC1_0571 (D3VIU5, yecC, ABC ATPase, PTHR43166) and XNC1_0572 (D3VIU6, yecS, "L-cystine transport system permease protein TcyL", PTHR30614)
Core Functions
Periplasmic substrate-binding subunit of an ABC importer, predicted to capture L-cystine in the periplasm and deliver it to the adjacent YecSC-type (TcyLN-type) permease and ATPase for ATP-driven uptake. Inferred from PANTHER subfamily membership with B. subtilis TcyA, conservation of the Ngo0372 cystine-pocket residues and operon context; ligand specificity is untested.
L-cystine-binding SBP function is therefore a reasonable inference, but it rests on subfamily membership with TcyA, conserved pocket residues and synteny, not on orthology to E. coli FliY.
Q: Does D3VIU4 bind L-cystine with nanomolar affinity like Ngo0372, or does it prefer a related sulfur amino acid (cystathionine, djenkolate, cysteine)?
Q: Which of the two adjacent SF34 binding proteins (D3VIU4/XNC1_0570 and D3VIU8/XNC1_0574) delivers substrate to the XNC1_0571-0572 permease/ATPase?
Suggested Experiments
Experiment: Isothermal titration calorimetry or intrinsic tryptophan fluorescence of purified mature D3VIU4 (residues 29-262) with L-cystine and related sulfur amino acids; test the Tyr49Ala mutant.
Experiment: Growth of X. nematophila XNC1_0570 and XNC1_0574 single and double deletions on L-cystine as sole sulfur source, with radiolabelled cystine uptake.
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
D3VIU4 (XNC1_0570, submitter name fliY) is a 262-residue protein with an N-terminal signal peptide and a single family-3 solute-binding domain, the architecture of a soluble periplasmic substrate-binding subunit of an ABC importer, most likely for L-cystine. It has no transmembrane segments, so it cannot form an ion-conducting pore, and the one ProtNLM2 prediction (ligand-gated monoatomic ion channel activity) is refuted by the domain architecture. No experimental study of D3VIU4 was found in PubMed.
Review score: 2 = concordant with evidence; 1 = uncertain; 0 = discordant with evidence. This is an assessment score, not a model probability.
GO:0015276 ligand-gated monoatomic ion channel activityGO_MF
NPI — Nonparalog incorrect Review score: 0/2
Prediction method: ProtNLM2 · Version: UniProt 2024_06 pilot
DOMAIN ARCHITECTURE MISMATCH
Review rationale: The 262-residue target has an N-terminal signal peptide (1-28) followed by a solute-binding protein family 3 domain covering residues 40-259, and the record has no transmembrane segments. This is the architecture of an exported periplasmic binding protein, not a pore-forming channel. Family-3 binding modules resemble the ligand-binding lobes of ionotropic glutamate receptors, but in those channels the module is fused to a transmembrane pore domain that D3VIU4 lacks. In the characterised E. coli cystine importer (FliY, a sister subfamily) the binding protein hands its ligand to separate membrane-spanning and ATPase partners (YecS, YecC), which is the role the gene review assigns to D3VIU4 alongside its neighbouring ATPase and permease genes. The gene review carries no channel annotation, the PANTHER PTHR35936 family review records that the target lacks the pore architecture required for this activity, and the OpenScientist investigation of this prediction reached the same conclusion from domain and structural analysis. Binding a transported ligand does not make the binding component a ligand-gated ion channel.
PMID:26350134: "inspection suggested that YecS and YecC were likely to be the ATPase and membrane-spanning partners of FliY in an ATP-binding cassette (ABC) transport system"