Scope. De-novo review for the CACAO audit project (projects/CACAO.md). Four of
the six GOA rows on P39234 are assigned_by: CACAO (IDA, 2012), all from two papers
about gp5, the product of the adjacent gene 5.
Deep research was unavailable for this gene. No deep-research API keys are
configured in this container, so just deep-research was not run and there is no
5.4-deep-research-*.md file. The literature work below was done directly: reading
the cached publications, PubMed/PMC via the PubMed MCP tools, and the RCSB entry for
PDB 4KU0. The GONUTS wiki (gowiki.tamu.edu), which would record the students'
evidence for the CACAO rows, was queried on 2026-10-29 for
PMID:3157805 and returned only "This site is temporarily offline while we perform
upgrades" — the same state projects/CACAO.md records, so the CACAO rows below are
judged from the papers and from the structural literature alone.
A 97-residue (10.2 kDa) protein of the PAAR-repeat superfamily that caps the tip of
the T4 central spike. UniProt:
Baseplate central spike complex-associated protein. 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.
[file:BPT4/5.4/5.4-uniprot.txt, CC:FUNCTION]
It sits in the baseplate as part of the central spike complex, one copy per spike:
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, CC:SUBUNIT]
The subcellular location is curated from the cryo-EM baseplate structure
(ECO:0000269|PubMed:27193680): "Virion", with "Note=Present in the baseplate."
Pfam assigns one PAAR_motif domain (DR Pfam; PF05488; PAAR_motif; 1.) and CDD
cd14737; PAAR_1.
gp5.4 was identified as the T4 PAAR protein by Shneider et al. 2013, who were looking
for the phage counterpart of the T6SS VgrG spike cap:
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 β-helical domain of
the central spike protein gp5
PMID:23925114Proteins containing the PAAR-repeat motif were strongly represented in this group
with gp5.4 being the corresponding protein of T4 phage.
PMID:23925114
That paper's crystal structures are of two bacterial PAAR proteins (VCA0105,
c1882) bound to a T4 gp5 β-helix fragment, and T4 gp5.4 features in it only as the
identified phage member and as the template for a surface-mimicking triple mutant
(c1882* contains three mutations T28K, T64K, T90K that were made to mimic T4
gp5.4.). Its functional conclusion is nonetheless the mechanistic basis for the
gp5.4 function statement:
Because a PAAR-repeat protein caps the end of the β-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.
PMID:23925114
The direct structural evidence for gp5.4 itself is PDB 4KU0 (Buth, Leiman &
Shneider, submitted May 2013; RP X-RAY CRYSTALLOGRAPHY (1.15 ANGSTROMS) OF 2-97 IN
COMPLEX WITH IRON, COFACTOR, AND FUNCTION.), titled "Enterobacteria phage T4 gp5.4
PAAR repeat protein in complex with T4 gp5 beta-helix fragment" (RCSB entry, checked
2026-10-29) — i.e. gp5.4 bound to the blunt end of the gp5 β-helix, exactly the
arrangement Shneider et al. inferred. It is an unpublished deposition, cited by
UniProt as Ref.2.
UniProt records a single Fe cation cofactor with four binding residues — His14,
His46, His56 and Cys83 (FT BINDING ... /ligand="Fe cation", evidence
ECO:0007744|PDB:4KU0) — and the RCSB ligand list for 4KU0 includes FE (Fe(III)),
plus Mg and Na. The site is the conserved PAAR metal site, but in the T6SS PAAR
proteins it was shown by X-ray fluorescence and anomalous difference maps to be zinc:
The fold of the PAAR protein is stabilized by a Zn atom positioned close to the
cone’s vertex
PMID:23925114The 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 residue positions line up with gp5.4 almost exactly (H14/H46/H56/C83 vs
H14/H46/H54/C81), so the site is clearly homologous; the identity of the ion in
gp5.4 rests on one unpublished 1.15 Å structure and is therefore not settled. The
role of the ion is structural:
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.
PMID:23925114
This is why the GOA row stays at GO:0046872 metal ion binding rather than being
refined to iron ion binding — the broad term is as precise as the evidence firmly
allows.
Four CACAO IDA rows (2012-10-30 to 2012-11-15) come from two papers, both of which
are about gp5, the 575-residue product of gene 5, and neither of which mentions
gene 5.4 — indeed the gp5.4 protein was an unnamed density in the 2003 baseplate
reconstruction and was not identified until 2013.
PMID:3157805 (Nakagawa, Arisaka & Ishii 1985) states the identification in its first
sentence:
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 enzyme they purified by isoelectric focusing has a mass of 42 kDa and
N-acetylmuramidase activity ("The molecular weight as determined from sodium dodecyl
sulfate electrophoresis was 42,000"; "The tail-lysozyme has N-acetylmuramidase
activity and the same substrate specificity as gene e lysozyme"). gp5.4 is 10.2 kDa.
PMID:10217762 (Kanamaru et al. 1999) is likewise explicit:
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.
PMID:10217762
Its subject is the cleavage of the 63 kDa gp5 precursor into mature gp5* and the
C-terminal fragment gp5C, and the finding that gp5C stays in the particle ("the
C-terminal fragment was found in the tail and the phage particle by immunoblotting").
"gp5C" is a cleavage product of P16009, not gene 5.4.
Consequences for the review:
GO:0003796 lysozyme activity (IDA, PMID:10217762) and GO:0033922 peptidoglycan
beta-N-acetylmuramidase activity (IDA, PMID:3157805) are REMOVE. gp5.4 has nosource_defects of type WRONG_GENE_PRODUCT (P39234 → P16009).GO:0098025 virus tail, baseplate CACAO rows are ACCEPT: the claim issource_defects entry asking for the reference toFailure-taxonomy note for projects/CACAO.md. This is the "one paper applied to a
gene it does not assay" shape, in a sharper form than the BACSU case: not a paper
covering several genes, but an adjacent-locus identifier confusion — "gp5" in the
papers resolved to the UniProt entry whose gene name is 5.4 instead of 5. It is
worth flagging separately because two of the four misattributed rows (the baseplate
CC rows) are accidentally true of gp5.4, so an inventory counting accepted rows
would score them as successes. Both wrong rows are molecular-function rows, where the
accident does not save them.