ACTL7A actin-fold audit: does the fold still carry actin's working parts?

Reproduce with uv run --script actin_fold_audit.py (regenerates results.json
and this file byte-for-byte; inputs are cached under data/, which is not committed).

Why

ACTL7A is annotated with actin-derived terms (GO:0005200 structural constituent of cytoskeleton, GO:0007010 cytoskeleton organization, GO:0005198 structural molecule activity). Those hold only if ACTL7A retains the machinery the terms imply. This audit
derives two residue sets from experimental actin structures and reports what ACTL7A has
at those positions, against a panel that spans the family from conventional actin to the
most divergent SWI/SNF Arps.

Findings

Whole-chain identity of ACTL7A to the deposited actin: 43.7%.
Nucleotide cleft largely retained. ACTL7A is 63.2% identical to actin across the 19 G-actin cleft positions (66.7% at the phosphate contacts), against 96.5% for conventional actins, 79.0% for Arp2/Arp3 and 36.9% for the divergent SWI/SNF Arps.
The ATP-hydrolysis trigger is not retained. Of the 5 literature-defined catalytic positions ACTL7A keeps 3 (DQEVY); changed: D154->E, H161->Y. Every conventional actin and Arp1/Arp2/Arp3 in the panel keeps all of them.
The filament interface is not retained. ACTL7A matches actin at 42.3% of the 79 inter-protomer contact positions, versus 93.2% for conventional actins and 55.7% for Arp1, which does form a filament (in dynactin).
The cleft is under disease-relevant constraint. 2/5 ACTL7A variants reported in SPGF86 patients map into the nucleotide cleft, which covers only 4.6% of the protein (binomial p = 0.0193); 0/4 population polymorphisms do.

Method

Nucleotide-cleft residues: every residue within 4.0 A of
ATP or the divalent cation in PDB 2BTF (profilin-beta-actin), and of ADP/Pi/Mg in
PDB 8A2S (cryo-EM F-actin, Mg-ADP-Pi, 5 protomers).
Inter-protomer interface: every residue within 4.5 A of a
different protomer in 8A2S, required in >=50% of the protomers that have any such contact
(so the terminal protomers do not dilute the set).
Alignment: mafft --localpair --maxiterate 1000 --anysymbol (v7.526 (2024/Apr/26)), with the deposited SEQRES sequences
included as alignment entries so that structural positions map without hard-coded numbering.
Contact sets are computed by neighbour search, never listed by hand.

Site sizes

site	n positions	description
`g_pocket_all`	19	any residue within 4.0 A of ATP or the divalent cation (PDB 2BTF (profilin-beta-actin, ATP))
`g_pocket_adenine`	4	within 4.0 A of the ATP adenine ring (PDB 2BTF)
`g_pocket_phosphate`	12	within 4.0 A of the ATP alpha/beta/gamma phosphates (PDB 2BTF)
`g_pocket_metal`	1	within 4.0 A of the divalent cation (PDB 2BTF)
`f_pocket_all`	20	within 4.0 A of ADP/Pi/Mg in >=50% of protomers (PDB 8A2S (cryo-EM F-actin, Mg-ADP-Pi))
`f_protomer_interface`	79	within 4.5 A of a neighbouring protomer in >=50% of protomers that have any inter-protomer contact (PDB 8A2S)

Conservation at the derived sites

% id = identity to the deposited actin sequence at that site's positions.

protein	group	len	% id vs actin (whole chain)	nucleotide cleft, G-actin	phosphates	metal	adenine	nucleotide cleft, F-actin	protomer interface
ACTL7A_HUMAN	query	435	43.7	63.2	66.7	100.0	50.0	60.0	42.3
ACTL7A_MOUSE	query_ortholog	440	43.7	63.2	66.7	100.0	50.0	60.0	41.0
ACTL7A_RAT	query_ortholog	440	44.2	63.2	66.7	100.0	50.0	60.0	41.0
ACTL7B_HUMAN	query_paralog	415	44.6	68.4	75.0	100.0	50.0	65.0	36.4
ACTL9_HUMAN	testis_arp	416	41.2	57.9	66.7	100.0	25.0	55.0	29.5
ACTL10_HUMAN	testis_arp	245	33.3	50.0	71.4	0.0	25.0	46.7	18.2
ACTRT1_HUMAN	testis_arp	376	48.7	73.7	83.3	0.0	50.0	75.0	39.7
ACTRT2_HUMAN	testis_arp	377	48.5	68.4	75.0	100.0	25.0	65.0	41.8
ACTRT3_HUMAN	testis_arp	372	49.2	78.9	91.7	100.0	50.0	70.0	43.6
ACTB_HUMAN	conventional_actin	375	99.7	100.0	100.0	100.0	100.0	95.0	96.2
ACTA1_HUMAN	conventional_actin	377	93.3	94.7	91.7	100.0	100.0	100.0	100.0
ACT1_YEAST	conventional_actin	375	88.5	94.7	100.0	100.0	75.0	90.0	83.5
ARP1_HUMAN	filament_forming_arp	376	52.8	73.7	83.3	100.0	50.0	70.0	55.7
ARP2_HUMAN	nucleotide_binding_arp	394	47.5	84.2	83.3	100.0	100.0	85.0	41.8
ARP3_HUMAN	nucleotide_binding_arp	418	38.7	73.7	75.0	100.0	75.0	70.0	24.4
ARP4_BAF53A_HUMAN	nuclear_arp	429	37.1	31.6	41.7	0.0	25.0	30.0	38.0
ARP5_HUMAN	nuclear_arp	607	29.2	42.1	41.7	0.0	75.0	40.0	22.4
ARP6_HUMAN	nuclear_arp	396	28.6	42.1	41.7	0.0	50.0	40.0	21.4
ARP8_HUMAN	nuclear_arp	624	22.1	52.6	50.0	0.0	75.0	50.0	17.7
ARP4_YEAST	nuclear_arp	489	32.9	47.4	41.7	0.0	50.0	45.0	29.2
ARP7_YEAST	divergent_swisnf_arp	477	22.9	42.1	41.7	0.0	0.0	40.0	16.7
ARP9_YEAST	divergent_swisnf_arp	467	18.7	31.6	33.3	0.0	25.0	30.0	20.3

Residue-by-residue at the G-actin nucleotide cleft

Positions (2BTF numbering): 13, 14, 15, 16, 18, 137, 156, 157, 158, 159, 182, 213, 214, 301, 302, 303, 305, 306, 336

protein	residues at those positions
2BTF actin (reference)	`GSGMKQGDGVGKEGGTMYK`
ACTL7A_HUMAN	`GTGYKQGHGVGKKGGSMLD`
ACTL7A_MOUSE	`GTGFKQGHGVGKTGGSMLD`
ACTL7A_RAT	`GTGFKQGHGVGKTGGSMLD`
ACTL7B_HUMAN	`GSQYKQGHGVGKKGGCMLK`
ACTL9_HUMAN	`GTGTKQGHGVGKHGGSLFN`
ACTL10_HUMAN	`-----TGAGVGKKGGSLFG`
ACTRT1_HUMAN	`GSGLKHGDGVGKEGGTLLC`
ACTRT2_HUMAN	`GSGFKQGDAVGKKGGTLFW`
ACTRT3_HUMAN	`GSGMKQGAGVGKEGGSSFK`
ACTB_HUMAN	`GSGMKQGDGVGKEGGTMYK`
ACTA1_HUMAN	`GSGLKQGDGVGKEGGTMYK`
ACT1_YEAST	`GSGMKQGDGVGKEGGTMFK`
ARP1_HUMAN	`GSGVKQGDGVGKEGGSLFL`
ARP2_HUMAN	`GTGFKQGDGVGKEGGSMYK`
ARP3_HUMAN	`GTGYKQGDGVGKEGGSMFR`
ARP4_BAF53A_HUMAN	`GSYTRTGATHGQAGGNLIR`
ARP5_HUMAN	`GSFQRDGYQCGLHGGNMYV`
ARP6_HUMAN	`GAYNKAGYSFGKEGGNLFI`
ARP8_HUMAN	`GSTTREGDQKGKEGGGMFR`
ARP4_YEAST	`GSYTNTGHDTGKEGGTSIQ`
ARP7_YEAST	`GSHRVEGASGGKSGSTLIK`
ARP9_YEAST	`RSQTLAGTHHGAKGGTSIS`

The ATP-hydrolysis catalytic set

Actin residues D11, Q137, D154, V159, H161 (standard actin numbering, verified against the 2BTF SEQRES).
Gln137 and His161 are the hydrolysis pair; Asp154 and the Val159 main chain stabilise the
attacking water; Asp11 is part of the divalent-cation site. Sources: PMID:37009486,
PMID:30622175.

protein	group	residues at D11, Q137, D154, V159, H161	conserved / 5
ACTL7A_HUMAN	query	`DQEVY`	3
ACTL7A_MOUSE	query_ortholog	`DQEVY`	3
ACTL7A_RAT	query_ortholog	`DQEVY`	3
ACTL7B_HUMAN	query_paralog	`DQEVH`	4
ACTL9_HUMAN	testis_arp	`DQDVY`	4
ACTL10_HUMAN	testis_arp	`-TEVH`	2
ACTRT1_HUMAN	testis_arp	`DHDVC`	3
ACTRT2_HUMAN	testis_arp	`DQDVC`	4
ACTRT3_HUMAN	testis_arp	`DQNVQ`	3
ACTB_HUMAN	conventional_actin	`DQDVH`	5
ACTA1_HUMAN	conventional_actin	`DQDVH`	5
ACT1_YEAST	conventional_actin	`DQDVH`	5
ARP1_HUMAN	filament_forming_arp	`DQDVH`	5
ARP2_HUMAN	nucleotide_binding_arp	`DQDVH`	5
ARP3_HUMAN	nucleotide_binding_arp	`DQDVH`	5
ARP4_BAF53A_HUMAN	nuclear_arp	`DTDHT`	2
ARP5_HUMAN	nuclear_arp	`DDSCH`	2
ARP6_HUMAN	nuclear_arp	`DADFH`	3
ARP8_HUMAN	nuclear_arp	`HEDKS`	1
ARP4_YEAST	nuclear_arp	`DTDTS`	2
ARP7_YEAST	divergent_swisnf_arp	`HEDGN`	1
ARP9_YEAST	divergent_swisnf_arp	`YADHD`	1

ACTL7A variants against the derived sites

ACTL7A numbering includes its 64-residue N-terminal extension, so ACTL7A position n
aligns to roughly actin position n - 64; the mapped actin position is given explicitly.

variant	reported in SPGF86	aligned actin position	actin residue there	falls in
R45C	no	-	-	-
D75A	yes	11	D	-
A161P	no	97	A	-
A245T	yes	181	A	-
G246A	yes	182	G	`f_pocket_all`, `g_pocket_all`
V340M	no	279	F	-
L343V	no	282	I	-
G362R	yes	301	G	`f_pocket_all`, `g_pocket_all`, `g_pocket_phosphate`
G402S	yes	342	G	-

20 of ACTL7A's 435 residues align to a nucleotide-cleft column (4.6% of the protein). 2/5 variants reported in SPGF86 patients fall there (exact binomial upper tail p = 0.0193), versus 0/4 population polymorphisms.

Caveat: n is small and the variant set is not an unbiased sample, so this is suggestive only; UniProt annotates A245T, G246A and G362R as 'uncertain significance'.

Alignment windows around every mapped position

Included so the mapping can be checked rather than trusted: if these windows were
gap-ridden the residue calls above would be alignment artefacts.

actin 3-19:

ACTB_2BTF_chainA     DDIAALVVDNGSGMCKA
ACTB_HUMAN           DDIAALVVDNGSGMCKA
ACTA1_HUMAN          DETTALVCDNGSGLVKA
ACTL7A_HUMAN         EVTKAVVVDLGTGYCKC
ACTL7B_HUMAN         HKIKAVIIDLGSQYCKC
ACTL9_HUMAN          PKTGAVVIDMGTGTCKV
ARP1_HUMAN           IANQPVVIDNGSGVIKA
ARP2_HUMAN           QGRKVVVCDNGTGFVKC
ARP9_YEAST           RQDSILIIYPRSQTTLV

actin 89-105:

ACTB_2BTF_chainA     TFY-NEL-RVAPE-----------EH-PVLL
ACTB_HUMAN           TFY-NEL-RVAPE-----------EH-PVLL
ACTA1_HUMAN          TFY-NEL-RVAPE-----------EH-PTLL
ACTL7A_HUMAN         LFR-QEM-KIAPE-----------EH-AVLV
ACTL7B_HUMAN         IFR-TAM-KILPE-----------EH-AVLV
ACTL9_HUMAN          LLE-HDL-RVATH-----------DH-PLLF
ARP1_HUMAN           VYSKDQL-QTFSE-----------EH-PVLL
ARP2_HUMAN           TFGPEKL-NIDTR-----------NC-KILL
ARP9_YEAST           IFV-SIL-SDRANKNQDAFEAELSNI-PLLL

actin 129-169:

ACTB_2BTF_chainA     TPAMYVAIQAVLSLYASGRT--------TGIVMDSGDGVTHTVPIYEGY
ACTB_HUMAN           TPAMYVAIQAVLSLYASGRT--------TGIVMDSGDGVTHTVPIYEGY
ACTA1_HUMAN          VPAMYVAIQAVLSLYASGRT--------TGIVLDSGDGVTHNVPIYEGY
ACTL7A_HUMAN         TPAMHIAYQSRLSMYSYGRT--------SGLVVEVGHGVSYVVPIYEGY
ACTL7B_HUMAN         IPAMHVTSQSLLSIYSYGKT--------SGLVVESGHGVSHVVPISEGD
ACTL9_HUMAN          SPAMYVASQSVLSVYAHGRV--------SGLVVDTGHGVTYTVPVFQGY
ARP1_HUMAN           VPALFISMQAVLSLYATGRT--------TGVVLDSGDGVTHAVPIYEGF
ARP2_HUMAN           FSGVYVAIQAVLTLYAQGLL--------TGVVVDSGDGVTHICPVYEGF
ARP9_YEAST           INNLIQLPASLAATYSMISL-------QNCCIIDVGTHHTDIIPIVDYA

actin 173-190:

ACTB_2BTF_chainA     HAILRLDLAGRDLTDYLM
ACTB_HUMAN           HAILRLDLAGRDLTDYLM
ACTA1_HUMAN          HAIMRLDLAGRDLTDYLM
ACTL7A_HUMAN         SITGRLDYAGSDLTAYLL
ACTL7B_HUMAN         GLTSRADYAGGDLTNYLM
ACTL9_HUMAN          HATERLDLAGNNLTAFLA
ARP1_HUMAN           HSIMRIDIAGRDVSRFLR
ARP2_HUMAN           HLTRRLDIAGRDITRYLI
ARP9_YEAST           HLVSSIPMGGQSINDSLK

actin 271-290:

ACTB_2BTF_chainA     SCGIHETTFNSIMKC-DVDIR
ACTB_HUMAN           SCGIHETTFNSIMKC-DVDIR
ACTA1_HUMAN          SAGIHETTYNSIMKC-DIDIR
ACTL7A_HUMAN         QLGLHTQTVSCLNKC-DIALK
ACTL7B_HUMAN         QPGLPELTAACLGRCQDTGFK
ACTL9_HUMAN          PVGLSTMAKQSLRKL-SLEMR
ARP1_HUMAN           SEGIHEVLVFAIQKS-DMDLR
ARP2_HUMAN           GVGVAELLFNTIQAA-DIDTR
ARP9_YEAST           -KNISNRVGLTLDNIDDINKA

actin 293-309:

ACTB_2BTF_chainA     LYANTVLSGGTTMYPGI
ACTB_HUMAN           LYANTVLSGGTTMYPGI
ACTA1_HUMAN          LYANNVMSGGTTMYPGI
ACTL7A_HUMAN         LMGNILLCGGSTMLSGF
ACTL7B_HUMAN         MAANVLLCGGCTMLDGF
ACTL9_HUMAN          LAQNVLLCGGSSLFTGF
ARP1_HUMAN           LFSNIVLSGGSTLFKGF
ARP2_HUMAN           FYKHIVLSGGSTMYPGL
ARP9_YEAST           VWENIIIVGGTTSISGF

actin 334-350:

ACTB_2BTF_chainA     ERKYSVWIGGSILA-SLS
ACTB_HUMAN           ERKYSVWIGGSILA-SLS
ACTA1_HUMAN          ERKYSVWIGGSILA-SLS
ACTL7A_HUMAN         ERDSAVWTGGSILA-SLQ
ACTL7B_HUMAN         ERKTSVWTGGSILA-SLQ
ACTL9_HUMAN          TRNFSVWIGGSILA-SLR
ARP1_HUMAN           ERLYSTWIGGSILA-SLD
ARP2_HUMAN           RRKHMVFLGGAVLA-DIM
ARP9_YEAST           GYSEIIFLGAQIVSKQIF

Notes on interpretation

This is a sequence-and-structure audit, not an assay. Retained residues mean the
activity is not excluded; they do not demonstrate it. Lost residues are the stronger
signal, because they exclude the activity on structural grounds.
The comparator groups are the yardstick: read ACTL7A's numbers against the conventional
actins (which bind nucleotide and polymerise) and against ARP7/ARP9 (the family's most
divergent members).
Percentages at a site are identity to this particular actin, so a conservative
substitution counts as a mismatch. The residue-level table above is the thing to read
when a percentage looks low.