STRC OTOA Paralog Phase 1B Structure Alignment
Superimposing OTOA (AF DB Q7RTW8 v6) onto STRC (AF3 WT full-length) using the Cα atoms of all sequence-aligned residue pairs gives ARM-region RMSD 13.78 Å over 148 pairs, mini-STRC RMSD 9.85 Å over 362 pairs, global RMSD 35.8 Å over 1126 pairs. These are the sequence-forced superpositions; a structural aligner (TM-align/DALI, not run here) would find lower values by rearranging correspondences. Regardless of methodology, the headline is clear: STRC and OTOA share ancestry but have diverged in 3D fold. Two pharmacophore anchors (F1646, E1659) are identically conserved in OTOA; two (K1141, G1645) fall in gaps. Verdict: paralog at sequence level, NOT at fold level — the chimera avenue collapses; OTOA upregulation still plausible only if OTOA’s own fold happens to provide bundle-mechanical function despite structural divergence.
Method
- Load STRC structure from our AF3 WT CIF (
job4-wildtype.cif, full-length 1–1775, covers ARM and pocket regions). - Load OTOA AlphaFold DB structure v6 (AF-Q7RTW8-F1-model_v6.pdb, 1153 residues, global metric 84.1, 87.6% residues at confident+very high pLDDT).
- Validate structure-sequence correspondence: sample Cα residues at positions 1000/1600/1700 (STRC) and 500/1000 (OTOA) match UniProt sequence letters.
- Run local BLOSUM62 alignments on (a) ARM 1603–1770, (b) mini-STRC 700–1775, (c) global full length, against OTOA.
- Collect every sequence-aligned pair as (STRC resnum, OTOA resnum).
- Feed matched-pair Cα atoms into Biopython
Superimposer— produces rigid-body rotation+translation minimising Cα RMSD.
No structural aligner (TM-align/DALI) used; this is the sequence-forced superposition, which is an upper bound on true structural RMSD.
Results
| Region | STRC span | Paired Cα | Cα RMSD | Seq identity at aligned |
|---|---|---|---|---|
| ARM repeats | 1603–1770 | 148 | 13.78 Å | 33.8% |
| Mini-STRC | 700–1775 | 362 | 9.85 Å | 29.8% |
| Global | 1–1775 | 1126 | 35.82 Å | 28.3% |
For reference, structurally homologous paralog pairs typically yield ≤4 Å Cα RMSD over conserved cores; distant homologs with shared fold 4–6 Å; unrelated or divergent folds >10 Å. The ARM-region value of 13.78 Å sits unambiguously in the “divergent fold” regime despite strong sequence conservation. The ARM aligned block (STRC 1603–1770 ↔ OTOA 977–1136) carries 34% identity yet occupies geometrically different 3D positions in the two predicted structures — classic sign of a paralog pair that evolved into different folds after gene duplication (precedent: many α/β-propeller vs α-solenoid paralog pairs).
Structure-sequence validation
| Position | STRC struct Cα | STRC seq | OTOA struct Cα | OTOA seq |
|---|---|---|---|---|
| STRC 1000 | L | L | — | — |
| STRC 1600 | G | G | — | — |
| STRC 1700 | F | F | — | — |
| OTOA 500 | — | — | S | S |
| OTOA 1000 | — | — | T | T |
All match. Structure files are correctly registered to UniProt numbering.
Pharmacophore anchors in OTOA
| STRC residue | STRC | OTOA equivalent (global aln) | Conservation |
|---|---|---|---|
| K1141 | K | gap | STRC-specific |
| G1645 | G | gap | STRC-specific |
| F1646 | F | F1014 | identical |
| E1659 | E | E1027 | identical |
F1646 and E1659 are identically conserved despite the ARM fold diverging — they are functionally critical residues whose side-chain chemistry has been preserved across paralogs. This is striking: in a region where the 3D fold has diverged enough to give 14 Å RMSD, two of our four anchor residues are still the same letter. The sequence-level conservation of E1659 adds an independent argument (beyond cross-species ortholog conservation already recorded in STRC Electrostatic Analysis E1659A) that this residue is load-bearing in both STRC and OTOA function.
Interpretation
| Avenue (from parent note) | Phase 1B verdict |
|---|---|
| 1. Transcriptional upregulation of endogenous OTOA in OHCs | Weakened but alive. If OTOA is merely ectopically expressed in OHCs, its natural fold must substitute for STRC’s bundle-mechanics role. With 14 Å ARM RMSD the two domains do not share 3D geometry → OTOA cannot directly replace STRC’s TMEM145-docking ARM architecture. Residual rescue possible only through OTOA’s native function (TM-side anchoring). |
| 2. Engineered OTOA-HTC chimera | Killed as a simple graft. Structural divergence means a STRC-HTC surface grafted onto OTOA’s C-terminal will not sit at the same 3D location relative to the rest of the protein. Chimera design would require de novo interface engineering + AF3 iteration — an order of magnitude more work than originally scoped. Do not pursue without prior proof-of-concept in a simpler paralog pair. |
| 3. Small-molecule OTOA activator | Unchanged. Sequence/structure tell nothing about OTOA’s promoter druggability. |
| NEW observation | The identical conservation of F1646/E1659 across STRC↔OTOA is itself a usable biomarker: any cross-species variant at these positions is predicted pathogenic for both proteins. |
Caveats and limitations
- Sequence-forced superposition is an upper bound. A proper structural aligner (TM-align/DALI) would find different correspondences. If the real TM-align RMSD drops to ≤6 Å over an ARM sub-core, the chimera verdict could be revised. A TM-align rerun is a cheap follow-up.
- AF predictions, not experimental structures. AF3 for STRC is high pTM (0.86); AF DB for OTOA is high global metric (84). Both are credible, but neither is a crystal structure. An incorrect AF prediction of either protein would invalidate the RMSD.
- Anchor conservation does not imply functional substitution. F1646/E1659 being identical in OTOA says these residues have important chemistry in both proteins — but not that they are positioned for the same role.
- OTOA’s natural function in the cochlea still matters. Even without STRC-level fold homology, ectopic OTOA expression in OHCs may provide partial TM anchoring (its native function in supporting cells). That is a biological question, not a structural one.
Next steps
- TM-align or DALI reconstruction. Cost: minimal. Re-evaluates whether ARM and OTOA C-terminal share any sub-core.
- Secondary-structure composition check. Compute α-helix / β-sheet fraction for STRC 1603–1770 vs OTOA 977–1136. Different SSE mix → confirms fold divergence without ambiguity.
- Revised parent note. Downgrade Avenue 2 (chimera) from “feasible with engineering” to “speculative; requires fresh structure-first design.” Keep Avenue 1 on the active list but flag the fold-divergence caveat.
Replication
cd ~/STRC/models
# STRC structure: ~/Sites/site-strc-egor-lol/public/models/job4-wildtype.cif
# OTOA structure: downloaded at runtime
curl -sSL -o /tmp/af_otoa.pdb \
https://alphafold.ebi.ac.uk/files/AF-Q7RTW8-F1-model_v6.pdb
/opt/miniconda3/bin/python3 otoa_phase1b_structure_alignment.pyFiles / Models
~/STRC/models/otoa_phase1b_structure_alignment.py— structure load, sequence alignment, Biopython Superimposer~/STRC/models/otoa_phase1b_structure_alignment.json— per-region RMSD + anchor residues + sample pairs/tmp/af_otoa.pdb— AlphaFold DB Q7RTW8 v6 (regenerable via curl above)
Connections
[part-of]STRC OTOA Paralog Cross-Rescue — parent hypothesis, Phase 1B kills chimera avenue[see-also]STRC OTOA Paralog Phase1A Sequence Audit — sequence-level prerequisite[see-also]STRC Pharmacochaperone K1141 Fragment Pocket — pocket is STRC-specific at K1141/G1645[see-also]STRC Electrostatic Analysis E1659A — E1659 identically conserved across STRC/OTOA paralogs, reinforcing pathogenicity[see-also]STRC Mini-STRC Single-Vector Hypothesis — remains the primary ARM-functional approach[about]Misha