id: P09130 gene_symbol: traD product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:83333 label: Escherichia coli K-12 description: >- TraD is the type IV coupling protein of the F plasmid, a 717-residue inner membrane protein that connects the relaxosome to the secretion apparatus during conjugative DNA transfer. It is defined by what it joins rather than by a reaction it performs on its own: the relaxosome processes plasmid DNA at the origin of transfer, and TraD delivers that nucleoprotein complex to the membrane-spanning machine that translocates it into the recipient cell. The protein assembles into a hexameric ring that forms the cytoplasmic face of the conjugative pore, and it contacts the relaxosome through its C-terminal tail, which binds the relaxosome component TraM in grooves on a TraM tetramer. That tail is also the specificity determinant: shortening it lets TraD serve a broader range of mobilizable plasmids while reducing transfer of F itself. It belongs to the coupling-protein family whose other members include TrwB of R388, TraG of RP4 and VirD4 of the Agrobacterium T-DNA system. reference_proteome_assessments: - reference_proteomes: - UP000000625 status: NO_SUPPORTED_COUNTERPART reviewed_on: '2026-10-19' reason: Retain P09130 as the primary Swiss-Prot accession. No significant reference-proteome hit was returned. The reviewed entry explicitly derives from F and related plasmids, whereas the selected reference proteome does not provide a supported counterpart in this search. This is compatible with accessory-plasmid content; it is not proof of absence from E. coli K12 generally. evidence: - type: PROTEOME_SEARCH description: MMseqs2 searched 4,403 sequences across UP000000625 at E <= 1e-5 and sensitivity 7.5, returning 0 hits. The search table and candidate FASTA are empty; no alignments were generated. The heuristic search does not establish genomic absence or unique locus identity. supported_by: - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: 'target_sequence_count: 4403' - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: 'candidate_count: 0' - type: PROTEOME_MEMBERSHIP description: The primary accession is outside every searched exact-taxon reference proteome. supported_by: - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: 'primary_in_any_reference_proteome: false' - type: RECORD_HISTORY description: 'Sampled UniSave observations: 11-NOV-2015, entry version 110.: no Proteomes cross-reference; 02-DEC-2020, entry version 128.: no Proteomes cross-reference; 27-NOV-2024, entry version 141.: no Proteomes cross-reference. These dated observations do not establish uninterrupted membership/absence, the exact change date, or UniProt''s motivation.' supported_by: - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: |- entry_date: 11-NOV-2015, entry version 110. proteomes: [] - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: |- entry_date: 02-DEC-2020, entry version 128. proteomes: [] - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: |- entry_date: 27-NOV-2024, entry version 141. proteomes: [] - type: CURATION_CONTEXT description: The primary UniProt entry records plasmid origin. Reference-proteome membership at species or strain level does not ensure coverage of every accessory plasmid. supported_by: - reference_id: file:ECOLI/traD/traD-record-evidence.yaml supporting_text: 'geneEncodingType: Plasmid' references: - id: file:ECOLI/traD/traD-record-evidence.yaml title: Compact archived record and sequence evidence for P09130 - 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: PMID:18717787 title: Structural basis of specific TraD-TraM recognition during F plasmid-mediated bacterial conjugation. findings: - statement: >- The TraD C-terminal peptide binds four symmetry-related grooves on a TraM tetramer, the first structural evidence of a relaxosome-coupling protein interaction. supporting_text: The structure reveals the TraD C-terminal peptide bound to each of four symmetry-related grooves on the surface of the TraM tetramer. - statement: >- The TraD-TraM interaction is specific and required for efficient F conjugation in vivo, which is the evidence behind the existing conjugation annotation. supporting_text: Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Cached record is abstract-only. The abstract also characterises TraD as a hexameric ring ATPase forming the cytoplasmic face of the conjugative pore, which is treated here as a traceable statement rather than as an assay reported in this paper. - id: PMID:9811665 title: The carboxyl terminus of protein TraD adds specificity and efficiency to F-plasmid conjugative transfer. findings: - statement: >- TraD belongs to the coupling-protein family that also contains TrwB of R388, TraG of RP4 and VirD4 of Agrobacterium, whose members share transmembrane domains and nucleotide-binding sequence signatures. supporting_text: Proteins encoded by these genes, which are termed the TraG protein family, show significant similarities among their amino acid sequences and share transmembrane domains and sequence signatures for nucleoside triphosphate binding - statement: >- The C-terminal tail is the specificity determinant: truncating it broadens the range of relaxosomes TraD will serve while reducing F transfer efficiency. supporting_text: The change in specificity was due to a loss of some amino acids in the carboxyl terminus of TraD that resulted in a broadening of the range of mobilizable relaxosomes at the expense of a decrease in the efficiency of F-plasmid transfer. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Full text available in the cache. The clearest statement of the coupling-protein concept and of the family correspondence used by the type IV secretion module. - id: PMID:9324263 title: The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro. findings: - statement: >- TraM binds TraD in vitro, demonstrated by overlay assay and affinity chromatography. This is the first evidence of the relaxosome contact later resolved structurally. supporting_text: the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography. reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- Cached record is abstract-only. This is the reference UniProt cites for the inner-membrane location of P09130. - id: PMID:15919815 title: TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase. findings: - statement: >- TrwB, the R388 coupling protein and a member of the same family as TraD, has a characterised DNA-dependent ATPase activity. This is the basis for inferring ATP hydrolysis for TraD. supporting_text: In this work, we characterize a DNA-dependent ATPase activity for TrwBDeltaN70. - statement: >- The soluble domain of TrwB crystallises as a hexamer with a central channel, matching the hexameric ring architecture attributed to TraD. supporting_text: TrwBDeltaN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- Cited for a family member, not for TraD itself. That scope is stated on the annotation it supports, which carries an ISS evidence code with Q04230 in the supporting entities. - id: PMID:11214325 title: The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. findings: - statement: >- TrwB recruits the relaxosome, the same role TraD performs for F, so the family correspondence is functional and not merely one of sequence. supporting_text: This large multimeric protein is responsible for recruiting the relaxosome DNA-protein complex reference_review: relevance: LOW correctness: VERIFIED review_notes: >- Background for the family inference. Establishes the ring-helicase and F1-ATPase resemblance of the coupling-protein fold. existing_annotations: - term: id: GO:0005886 label: plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0037 review: summary: >- Correct. Unlike many annotations of this class, the underlying location is experimentally supported rather than only inferred from the subcellular-location vocabulary: UniProt cites PMID:9324263 for the inner membrane assignment, and TraD is an integral membrane protein, not a peripheral one. action: ACCEPT reason: >- The term matches the experimentally supported location. No more specific child is warranted, and the hexameric assembly at the cytoplasmic face of the pore is captured by the complex annotation instead. - term: id: GO:0009291 label: unidirectional conjugation evidence_type: IMP original_reference_id: PMID:18717787 qualifier: involved_in review: summary: >- Well supported. Mutations in the TraD-TraM interface are specific and required for efficient F conjugation in vivo, so the phenotype bears directly on TraD's contribution to conjugation rather than on a downstream consequence. action: ACCEPT reason: >- This is the right process term for a coupling protein, and the mutant evidence is specific to the interaction surface rather than to loss of the whole protein, which makes it unusually well targeted. Retained as a core annotation. - term: id: GO:0016887 label: ATP hydrolysis activity evidence_type: ISS original_reference_id: PMID:15919815 qualifier: enables supporting_entities: - UniProtKB:Q04230 review: summary: >- Proposed from the characterised family member rather than from an assay of TraD. TrwB, the R388 coupling protein, is a DNA-dependent ATPase that crystallises as a hexamer with a central channel, and it performs the same relaxosome-recruitment role for its plasmid that TraD performs for F. action: NEW reason: >- The evidence code is ISS on purpose and the supporting entity names the family member the inference comes from, so the basis stays explicit rather than being laundered into an apparent direct observation. No assay of F TraD's own nucleotide binding or hydrolysis was found. The inference is stronger here than the comparable one for F TraC, which is why a molecular function is proposed for this protein and withheld for that one: the coupling-protein family has a biochemically characterised member whose functional role matches TraD's, and the hexameric architecture is shared. - term: id: GO:0043684 label: type IV secretion system complex evidence_type: TAS original_reference_id: PMID:18717787 qualifier: part_of review: summary: >- TraD forms the cytoplasmic face of the conjugative pore, the entry point of the F-type secretion apparatus. UniProt independently records that it may form a hexamer and that it couples the transferosome to the type IV secretion system. action: NEW reason: >- Recorded with a traceable-author-statement code because the cited paper asserts the architecture as established background rather than determining the assembled machine itself; its own structure covers the TraD-TraM interface. Complex membership is nonetheless not in doubt, and GOA carries no cellular component for this protein beyond the membrane. - term: id: GO:0044097 label: secretion by the type IV secretion system evidence_type: IMP original_reference_id: PMID:9811665 qualifier: involved_in review: summary: >- C-terminal truncations of TraD change which relaxosomes it will serve and reduce the efficiency of F transfer, which is a direct mutant-phenotype demonstration that TraD acts in substrate delivery to the secretion system. action: NEW reason: >- The cargo-agnostic process term is proposed alongside, not instead of, unidirectional conjugation. The two capture different things: conjugation is the outcome for the plasmid, while this term is what TraD contributes to mechanistically, and the specificity-switching phenotype is evidence about the secretion step rather than about transfer success as such. core_functions: - description: >- Type IV coupling protein of the F plasmid. TraD assembles into a hexameric ring in the inner membrane that forms the cytoplasmic face of the conjugative pore, and it recruits the relaxosome to that pore by binding the relaxosome component TraM through its C-terminal tail. The same tail sets which relaxosomes the protein will accept, so TraD is the component that makes the otherwise generic secretion apparatus specific to its own plasmid. ATP hydrolysis is inferred from the characterised family member TrwB rather than assayed for TraD itself. molecular_function: id: GO:0016887 label: ATP hydrolysis activity directly_involved_in: - id: GO:0044097 label: secretion by the type IV secretion system - id: GO:0009291 label: unidirectional conjugation in_complex: id: GO:0043684 label: type IV secretion system complex locations: - id: GO:0005886 label: plasma membrane supported_by: - reference_id: PMID:18717787 supporting_text: Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. - reference_id: PMID:9811665 supporting_text: The change in specificity was due to a loss of some amino acids in the carboxyl terminus of TraD that resulted in a broadening of the range of mobilizable relaxosomes at the expense of a decrease in the efficiency of F-plasmid transfer. - reference_id: PMID:15919815 supporting_text: In this work, we characterize a DNA-dependent ATPase activity for TrwBDeltaN70. knowledge_gaps: - gap_statement: >- ATP hydrolysis by F TraD has not been demonstrated directly. The inference rests on TrwB, which is well characterised, but the two proteins differ in their C-terminal specificity regions and no F-specific kinetics exist. boundary: >- Keep the ISS evidence code and the supporting entity on the annotation so the inference is visible, and do not upgrade it without an assay of TraD. gap_kind: - BIOLOGY proposed_new_terms: - proposed_name: type IV secretion coupling protein activity proposed_definition: >- Enables the selective recruitment of a nucleoprotein complex to a type IV secretion apparatus, determining which substrates that apparatus accepts. justification: >- No existing molecular function expresses the coupling-protein role, which is the recruitment of one complex to another with plasmid-level selectivity. ATP hydrolysis is the activity the coupling protein enables, but it does not capture what the protein is for, and the C-terminal specificity determinant has no representation at all. The role is shared across a defined family whose members include TraD of F, TrwB of R388, TraG of RP4 and VirD4 of the Agrobacterium T-DNA system, so a term would serve more than one gene. Pending such a term, this review asserts ATP hydrolysis as the molecular function and records the recruitment and specificity roles in the description and process terms, rather than reaching for a binding term that would name the partner without naming the function. proposed_parent: id: GO:0140597 label: protein carrier activity supported_by: - reference_id: PMID:9811665 supporting_text: Proteins encoded by these genes, which are termed the TraG protein family, show significant similarities among their amino acid sequences and share transmembrane domains and sequence signatures for nucleoside triphosphate binding - reference_id: PMID:9811665 supporting_text: The change in specificity was due to a loss of some amino acids in the carboxyl terminus of TraD that resulted in a broadening of the range of mobilizable relaxosomes at the expense of a decrease in the efficiency of F-plasmid transfer. suggested_questions: - question: >- Can a molecular function be defined for coupling-protein activity? TraD, TrwB, TraG and VirD4 share a role that no current GO term expresses: they recruit a specific nucleoprotein complex to a translocation apparatus. ATP hydrolysis is what they enable, not what distinguishes them. - question: >- Does F TraD hydrolyse ATP, and is the activity DNA-dependent as it is for TrwB? The inference is standard but has not been tested for this protein. - question: >- Should the TraD-TraI interaction be curated alongside TraD-TraM? UniProt and the literature both note an in vivo TraD-TraI contact, but only the TraM interaction is structurally resolved and annotated. suggested_experiments: - description: >- Assay purified F TraD, or its soluble cytoplasmic domain, for ATP binding and hydrolysis in the presence and absence of single-stranded DNA, following the design used for the TrwB soluble domain. This would convert the ISS annotation proposed here into direct evidence or refute it. - description: >- Determine whether the C-terminal specificity tail affects ATP hydrolysis as well as relaxosome selection, by comparing the activity of full-length TraD with the truncations that broaden relaxosome range. A coupling between selection and catalysis would explain why broadened specificity costs F transfer efficiency.