Baseplate puncturing device gp5.4 of bacteriophage T4, a 97-residue (10.2 kDa) protein of the PAAR-repeat superfamily that forms the sharp conical tip of the virion central spike. A single copy of gp5.4 caps the blunt distal end of the trimeric C-terminal beta-helix of gp5, completing the central spike complex at the hub of the contractile tail baseplate together with the gp5 and gp27 trimers. Its small cone-shaped fold is stabilised by one metal ion held close to the vertex by three histidines and a cysteine (His14, His46, His56, Cys83); the ion is modelled as Fe(III) in the gp5.4-gp5 crystal structure, while the equivalent, sequence-conserved site of bacterial PAAR proteins carries zinc. Because it caps the spike, gp5.4 is the leading point of the injection machine and is thought to make the initial opening in the host outer membrane when sheath contraction drives the spike into the cell envelope - the step that precedes local peptidoglycan digestion by the gp5 lysozyme domain and ejection of the viral genome through the tail tube. gp5.4 is the phage counterpart of the PAAR proteins that sharpen the VgrG spike of the bacterial type VI secretion system.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003796 lysozyme activity | IDA PMID:10217762 The C-terminal fragment of the precursor tail lysozyme of ba... | REMOVE | Summary: CACAO row. The cited paper is about gp5, the 575-residue product of the adjacent gene 5: it measures the lysozyme activity of the gp5 precursor, of the nicked precursor and of mature gp5, and traces the fate of the cleaved C-terminal fragment gp5C. gp5.4 is a 97-residue PAAR-repeat protein with no glycoside hydrolase domain, and the paper does not mention it. Reason: The assay is on the right protein family but the wrong gene product. The abstract states in its first sentence that the tail lysozyme "is the product of gene 5 (gp 5)", and the masses it reports (63 kDa precursor, 43 kDa mature) are those of gp5, not of the 10.2 kDa gp5.4. Independently of the paper, gp5.4 cannot have this activity: the entry is 97 residues long with a single PAAR_motif domain (Pfam PF05488) and its structure is solved to 1.15 A as a metal-stabilised cone with no catalytic cleft, so there is no muramidase domain to carry EC 3.2.1.17. Nothing is lost by removal, because P16009 (gene 5) already carries lysozyme activity from this same reference. Note also that gp5.4 was an unidentified density in the 2003 baseplate reconstruction and was only recognised as a protein of the PAAR family in 2013 (PMID:23925114), fourteen years after this paper, so it cannot have been the subject of the assay. Supporting Evidence: PMID:10217762 Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. file:BPT4/5.4/5.4-uniprot.txt Pfam; PF05488; PAAR_motif; 1. PMID:23925114 Proteins containing the PAAR-repeat motif were strongly represented in this group with gp5.4 being the corresponding protein of T4 phage. |
| GO:0033922 peptidoglycan beta-N-acetylmuramidase activity | IDA PMID:3157805 Isolation and characterization of the bacteriophage T4 tail-... | REMOVE | Summary: CACAO row. The cited paper purifies the T4 tail-associated lysozyme by preparative isoelectric focusing, identifies it by two-dimensional gel electrophoresis as gp5, and shows it has N-acetylmuramidase activity with the same substrate specificity as the gene e lysozyme. The protein assayed has a mass of 42 kDa; gp5.4 is 10.2 kDa. Reason: Same misattribution as the GO:0003796 row above, and the term is the same activity (GO:0033922 is hydrolysis of terminal, non-reducing N-acetylmuramic residues, EC 3.2.1.17-equivalent chemistry). The abstract explicitly names gp5 as the enzyme and gives its mass as 42,000, which excludes the 97-residue gp5.4; gp5.4 has only a PAAR_motif domain and no catalytic cleft. P16009 (gene 5) carries lysozyme activity from this same paper, so the claim is not lost. Supporting Evidence: PMID:3157805 Direct evidence has been obtained that the tail-associated lysozyme of bacteriophage T4 (tail-lysozyme) is gp5, which is a protein component of the hub of the baseplate. PMID:3157805 The molecular weight as determined from sodium dodecyl sulfate electrophoresis was 42,000. file:BPT4/5.4/5.4-uniprot.txt Pfam; PF05488; PAAR_motif; 1. |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword-derived annotation that matches real structural evidence: the 1.15 A crystal structure of gp5.4 bound to the gp5 beta-helix fragment contains a single metal ion coordinated by His14, His46, His56 and Cys83, the conserved PAAR metal site, which stabilises the pointed vertex of the cone. Reason: The claim is correct and the general term is the right granularity here. UniProt curates an Fe cation cofactor for gp5.4 with four binding residues from PDB 4KU0, but the homologous site in the structurally characterised bacterial PAAR proteins was shown by X-ray fluorescence and anomalous difference maps to hold zinc, at residues that align with gp5.4's (H14/H46/H54/C81 in VCA0105 vs H14/H46/H56/C83 in gp5.4). Since the identity of the ion in gp5.4 rests on a single unpublished deposition, GO:0046872 is as specific as the evidence firmly allows and is not modified to GO:0005506 iron ion binding; if the Fe assignment is confirmed spectroscopically, the row should be refined then. The function of the ion is structural - it holds the tip of the cone together - so this is retained as part of the gene's core, puncturing-tip role rather than as a peripheral binding term. Supporting Evidence: file:BPT4/5.4/5.4-uniprot.txt Name=Fe cation; Xref=ChEBI:CHEBI:24875; file:BPT4/5.4/5.4-uniprot.txt X-RAY CRYSTALLOGRAPHY (1.15 ANGSTROMS) OF 2-97 IN COMPLEX WITH IRON, COFACTOR, AND FUNCTION. PMID:23925114 The Zn binding site consists of three histidines and one cysteine - H14, H46, H54 and C81 in VCA0105 - that are very well conserved in close homologs PMID:23925114 The metal ion, being a natural ligand for this site, stabilizes the pointed tip of the PAAR domain and is likely to be important for its integrity during penetration of the target cell envelope. |
| GO:0098025 virus tail, baseplate | IDA PMID:10217762 The C-terminal fragment of the precursor tail lysozyme of ba... | ACCEPT | Summary: CACAO row. The term is correct for gp5.4, which is a structural component of the baseplate central spike complex, but the cited paper establishes baseplate residence for gp5 and for its cleaved C-terminal fragment gp5C ("the C-terminal fragment was found in the tail and the phage particle by immunoblotting"), not for the product of gene 5.4. Reason: Kept, and the curator deferred to, because the claim is true and independently established by direct evidence: the cryo-EM structure of the baseplate places gp5.4 in the central spike complex (PMID:27193680, the row below), UniProt curates "Present in the baseplate" from that work, and PDB 4KU0 is the gp5.4 cone bound to the tip of the gp5 beta-helix. Removing a true cellular-component term that two other rows in this review also carry would be wrong. What is wrong is the reference-to-gene pairing, which is recorded as a source defect: a likely confusion of the cleavage product "gp5C" and the separate gene product "gp5.4", or of the UniProt entries named "5" and "5.4". The cached record is abstract-only (full_text_available: false) and the GONUTS wiki page that would hold the student's evidence was offline when queried on 2026-10-29 ("This site is temporarily offline while we perform upgrades"), so the figure-level reading could not be checked. Supporting Evidence: file:BPT4/5.4/5.4-uniprot.txt Note=Present in the baseplate. file:BPT4/5.4/5.4-uniprot.txt Part of the baseplate macromolecular complex which consists of gp5, gp5.4, gp27 (central spike complex); gp6, gp25, gp53 (inner baseplate); gp7, gp8 (intermediate baseplate); gp9, gp10, gp11, gp12 (peripheral); gp48 and gp54 (proximal region of the tail tube). |
| GO:0098025 virus tail, baseplate | IDA PMID:27193680 Structure of the T4 baseplate and its function in triggering... | ACCEPT | Summary: Near-atomic cryo-EM reconstruction of the 6 MDa T4 baseplate in its pre- and post-attachment states, from which gp5.4 was identified and modelled as part of the central spike complex; this is the direct evidence for the gene's location. Reason: Taylor et al. (2016) resolved the complete baseplate and assigned gp5.4 a position in the central spike complex with gp5 and gp27, which is what UniProt curates as the entry's subcellular location and subunit composition with ECO:0000269 to this paper. The term is at the right granularity: the baseplate is the structure gp5.4 belongs to, and the finer "tip of the central spike" placement has no cellular-component term. Supporting Evidence: PMID:27193680 Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. file:BPT4/5.4/5.4-uniprot.txt Note=Present in the baseplate. file:BPT4/5.4/5.4-uniprot.txt Heteromultimer with gp27 trimer and gp5 trimer; this interaction forms the tail lysozyme complex that creates an extension of the tail tube |
| GO:0098025 virus tail, baseplate | IDA PMID:3157805 Isolation and characterization of the bacteriophage T4 tail-... | ACCEPT | Summary: CACAO row. The term is correct for gp5.4, but the cited paper's baseplate-component claim is about gp5, the 42 kDa tail lysozyme it purified and identified by two-dimensional gel electrophoresis; gene 5.4 is not mentioned. Reason: Accepted for the same reasons as the PMID:10217762 baseplate row: the claim is true of gp5.4 and directly supported by the cryo-EM structure and the UniProt-curated location, and a true component term carried by three rows of this review must not be removed as a way of correcting one row's reference. The mis-pairing of reference and gene is recorded as a source defect. The cached record is abstract-only and GONUTS was offline on 2026-10-29, so the student's underlying reading could not be inspected; the abstract itself, however, attributes the baseplate hub component explicitly to gp5. Supporting Evidence: file:BPT4/5.4/5.4-uniprot.txt Note=Present in the baseplate. PMID:23925114 The cryoEM reconstruction of phage T4 baseplate shows that an unknown protein with a molecular weight between 7 and 23 kDa binds to the tip of the Ξ²-helical domain of the central spike protein gp5 |
| GO:0039660 structural constituent of virion | IDA PMID:27193680 Structure of the T4 baseplate and its function in triggering... | NEW | Summary: gp5.4 is a structural subunit of the virion: one copy caps the distal tip of each gp5 beta-helix in the baseplate central spike complex, completing the puncturing device of the contractile tail. Reason: The gene has no molecular function annotation describing what it actually does, which is to contribute to the structural integrity of the virion spike. This is established by direct structural evidence: the cryo-EM baseplate reconstruction assigns gp5.4 to the central spike complex, PDB 4KU0 is the 1.15 A structure of the gp5.4 cone bound to the gp5 beta-helix fragment, and UniProt lists gp5.4 as one of the three components of the central spike complex. gp5.4 itself does the work here - it is the physical tip, not an assembly factor or a substrate - and the same term was added on the same evidence to the other structural baseplate proteins of this phage (gp10, gp11, gp12). Supporting Evidence: file:BPT4/5.4/5.4-uniprot.txt Part of the baseplate macromolecular complex which consists of gp5, gp5.4, gp27 (central spike complex); gp6, gp25, gp53 (inner baseplate); gp7, gp8 (intermediate baseplate); gp9, gp10, gp11, gp12 (peripheral); gp48 and gp54 (proximal region of the tail tube). file:BPT4/5.4/5.4-uniprot.txt Baseplate central spike complex-associated protein. Makes the tip of the central spike PMID:23925114 Proteins containing the PAAR-repeat motif were strongly represented in this group with gp5.4 being the corresponding protein of T4 phage. |
| GO:0098994 symbiont entry into host cell via disruption of host cell envelope | IC PMID:23925114 PAAR-repeat proteins sharpen and diversify the type VI secre... | NEW | Summary: As the capping cone at the apex of the central spike, gp5.4 is the leading structure driven into the host envelope when the tail sheath contracts, and is held to make the initial opening in the outer membrane through which the spike and then the tail tube pass. Reason: Inferred from the structural placement of gp5.4 and from the general conclusion drawn for the PAAR superfamily, of which gp5.4 is the T4 member: because a PAAR protein caps the end of the spike beta-helix, it is the piercing tip responsible for creating the opening in the target cell envelope. gp5.4 therefore performs part of the work of envelope disruption itself - it is the physical point of the needle - rather than merely being required for it. The sibling term GO:0098932 (entry via disruption of host cell wall peptidoglycan) is not proposed, because the peptidoglycan step is catalysed by the gp5 lysozyme domain, not by gp5.4. The inference is flagged as such: UniProt states the puncturing role with ECO:0000305, and no gene 5.4 mutant has been assayed, so the proposal is made with IC rather than an experimental code. Supporting Evidence: PMID:23925114 Because a PAAR-repeat protein caps the end of the Ξ²-helix of a VgrG spike, it is in fact the piercing tip that is responsible for the initial event of creating an opening in the target cell envelope. file:BPT4/5.4/5.4-uniprot.txt Makes the tip of the central spike which probably punctures the host outer membrane, a necessary step for viral DNA ejection into the host cell. PMID:23925114 The metal ion, being a natural ligand for this site, stabilizes the pointed tip of the PAAR domain and is likely to be important for its integrity during penetration of the target cell envelope. |
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Download this section (compressed HTML)Q: Is the metal ion at the apex of gp5.4 iron or zinc? PDB 4KU0 models Fe(III) and UniProt curates an Fe cofactor, but every structurally characterised PAAR homologue carries Zn at the aligned 3-His/1-Cys site, verified by X-ray fluorescence. The answer decides whether GO:0046872 should be refined to GO:0005506 iron ion binding or to GO:0008270 zinc ion binding.
Q: Is gene 5.4 essential for infectivity, and is a spike without its PAAR cap still able to puncture the outer membrane? No amber or deletion mutant of gene 5.4 appears in the literature, although inactivating all PAAR genes severely impairs the analogous type VI secretion systems.
Q: Should GO have a cellular-component term for the tip of a contractile injection spike, or a molecular-function term for a spike-sharpening cap? gp5.4, the T6SS PAAR proteins and the DUF4280/PAAR-like proteins occupy the same structural position, and at present the only shared annotation available is the whole baseplate.
Q: Does gp5.4 stay attached to the spike after penetration, or does it dissociate like the gp5C beta-helix? This determines whether it is carried into the periplasm during genome delivery and whether it has any role after the initial opening is made.
Experiment: Determine the metal identity in gp5.4 directly: X-ray fluorescence scans and anomalous difference maps on gp5.4-gp5 crystals at the Fe and Zn edges, together with ICP-MS on purified recombinant gp5.4 and on intact T4 virions.
Hypothesis: The gp5.4 apex site holds zinc, as in all structurally characterised PAAR homologues, and the Fe(III) modelled in PDB 4KU0 is a crystallisation artefact or a mis-assignment of the density.
Experiment: Construct a gene 5.4 amber or deletion mutant (with a suppressor host or plasmid-borne complementation) and compare the resulting particles with wild-type for plaque-forming efficiency, adsorption, outer-membrane permeabilisation, and the kinetics of DNA ejection into cells and into liposome targets.
Hypothesis: Particles lacking gp5.4 assemble and adsorb normally but are defective in the initial penetration of the outer membrane, so genome ejection is delayed or abolished without the capping cone.
Experiment: Mutate the four metal-coordinating residues of gp5.4 (His14, His46, His56, Cys83) singly and in combination, and assay fold stability of the recombinant protein, its binding to the gp5 beta-helix fragment, its incorporation into virions, and infectivity of the resulting phage.
Hypothesis: The metal site is required for the integrity of the cone tip, so its loss leaves a protein that still binds the spike but fails during penetration, separating the capping and the puncturing contributions of gp5.4.
Experiment: Cryo-electron tomography of T4 particles caught attacking E. coli minicells or liposomes, with gp5.4 located by labelling or by difference mapping against a gene 5.4 deletion, imaging the spike before and after outer-membrane penetration.
Hypothesis: gp5.4 leads the spike through the outer membrane and then dissociates with the gp5C beta-helix, so it is not retained at the inner membrane during genome translocation.
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