Naked mole-rat cGAS (A0AAX6RS70): conservation of catalytic and chromatin-binding residues

Why this analysis was run

Every GO annotation on naked mole-rat cGAS is a TreeGrafter propagation from the
PANTHER ancestral node PTN000069395 (GO_REF:0000118); there is no experimental
GO annotation on this protein. Two of the propagated claims needed independent
checking before they could be accepted or rejected:

  1. 2',3'-cyclic GMP-AMP synthase activity (GO:0061501) — is the catalytic
    machinery intact in the naked mole-rat protein, or has it degenerated?
  2. negative regulation of DSB repair via homologous recombination (GO:2000042) —
    the primary literature reports that the naked mole-rat protein has lost this
    suppressive activity through changes at four amino acids, but the abstract does
    not name the residues. Can the reported positions be located and checked?

Method

align_cgas.py performs a global Needleman-Wunsch alignment (BLOSUM62,
gap open -11, extend -1, free end gaps) of naked mole-rat cGAS (UniProt
A0AAX6RS70, 554 aa) against human cGAS (Q8N884, 522 aa) and mouse cGAS
(Q8C6L5, 507 aa). It then parses every ACT_SITE, BINDING, and SITE feature
out of the reference UniProt flat files and transfers each position through the
alignment, reporting whether the naked mole-rat residue is identical or
substituted. No residue identity is hardcoded; all come from the records in
data/.

Reproduce with:

uv run python align_cgas.py

Full output is in alignment_output.txt.

Result 1: the catalytic core is fully intact

Global identity to human cGAS is 58.2% over aligned columns (55.2% to mouse) —
ordinary orthologue-level divergence. Every catalytically important residue that
UniProt annotates on human cGAS is present and identical in the naked mole-rat
protein:

function human mouse naked mole-rat conserved
catalytic Mg(2+) / ATP E225 E211 E257 yes
catalytic Mg(2+) / 2',3'-cGAMP D227 D213 D259 yes
catalytic Mg(2+) / GTP / 2',3'-cGAMP D319 D307 D351 yes
2',3'-cGAMP binding K362 K350 K394 yes
2',3'-cGAMP binding R376 — R408 yes
Zn(2+) thumb H390 H378 H422 yes
Zn(2+) thumb C396 C384 C428 yes
Zn(2+) thumb C397 C385 C429 yes
Zn(2+) thumb C404 C392 C436 yes
ATP binding S213 / K414 S199 / K402 S245 / K446 yes
nucleosome acidic-patch arginine anchor R255 R241 R287 yes

Against the human record, 12 of 15 annotated single-residue sites are identical
and 0 are gapped. The three substitutions are not catalytic: human T211
(GTP contact) → A243, and human K187/L195, which UniProt annotates as
"important for preferential detection of curved long DNA", → N220/Q228 in the
disordered/DNA-binding N-terminal arm, the least conserved part of the protein
in any mammalian comparison. Against the mouse record, 13 of 14 sites are
identical (only the same T→A).

Interpretation. The nucleotidyltransferase triad, the zinc thumb that
recognises B-form dsDNA, and the ATP/GTP/cGAMP contacts are all retained. There
is no pseudoenzyme signature. The catalytic-domain-level block identities are
also normal (62.5% over the C-terminal DNA-binding region 384-407; 69.0% over
341-382). Nothing in the sequence argues against the propagated
2',3'-cGAMP synthase activity, dsDNA binding, or nucleosome/chromatin binding.
The conserved arginine anchor (R287) is particularly relevant: this is the
residue that docks cGAS onto the nucleosome acidic patch, and its retention is
consistent with the reported chromatin retention of the naked mole-rat protein.

Result 2: the four divergent residues are C-terminal and regulatory, not catalytic

Chen et al. 2025 (PMID:41066557) state in the abstract only that the loss of HR
suppression happens "through the alteration of four amino acids during
evolution", without naming them. Secondary coverage of the paper names the four
substitutions as S463D, E511K, Y527L, T530K but does not state the numbering
frame. This analysis resolves that frame from sequence alone.

Reading the positions in naked mole-rat numbering gives an exact match to
the reported wild-type residues, and the aligned human and mouse residues are
exactly the mutant residues reported:

naked mole-rat aligned human aligned mouse
S463 D431 D416
E511 K479 K464
Y527 L495 L480
T530 K498 R483

So "S463D, E511K, Y527L, T530K" is a humanising substitution series applied
to the naked mole-rat protein: each mutation replaces the naked mole-rat residue
with the residue found at the aligned position in human (and, for three of four,
mouse) cGAS. This is internally consistent with the abstract's account of four
amino acids altered during evolution, and it was confirmed here from primary
sequence data rather than taken on trust from the secondary summary.

All four positions fall inside the C-terminal Mab-21-like HhH/H2TH-like domain
that UniProt annotates at 437-541 on A0AAX6RS70. All four are downstream of the
zinc thumb (422-436) and far from the catalytic triad (257/259/351).

Interpretation. The divergence that reverses the HR phenotype sits in a
C-terminal regulatory surface, not in the active site. This supports treating
the naked mole-rat protein as an enzyme with conserved catalysis whose
regulatory behaviour on chromatin has changed — consistent with the reported
mechanism (weakened TRIM41-mediated ubiquitination and weakened p97 interaction,
hence longer chromatin retention) — rather than as a protein that has lost or
swapped its molecular function.

Limits