STRC Research

STRC Mini-STRC Truncation Interface Validation

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STRC Mini-STRC Truncation Interface Validation

Structural superposition proof that the TMEM145 binding interface (canonical residues 1603-1749, C-terminal ARM repeats) is preserved across three STRC truncation candidates, including the aggressive C-terminal-only construct. Opens Ultra-Mini-STRC (1075-1775) as a viable therapeutic design with ~2.6 kb AAV headroom — sufficient for OHC-specific promoter + full regulatory cassette.

Question

The Derstroff et al 2026 TMEM145 Paper confirmed STRC binds TMEM145 via C-terminal ARM repeats. Prior AF3 extraction (strc_tmem145_interface_from_cif.json) mapped the interface to canonical residues 1603-1770 in Job 2 (mini-STRC 594-1775 × TMEM145).

But the actual clinical construct is 700-1775 (IgK-SP, CpG-depleted, see STRC CpG Depletion Mini-STRC). And the most aggressive truncation on the table is 1075-1775 (C-term only, job-h-strc-cterm-only.cif, pTM 0.87). Neither has been directly validated for interface preservation.

Question: Do the 700-1775 and 1075-1775 truncations preserve the C-terminal ARM fold responsible for TMEM145 anchoring?

If yes for both → Ultra-Mini-STRC (1075-1775) is therapeutically viable and opens a 2.6 kb AAV headroom (vs 1.5 kb for 700-1775), enabling OHC-specific Prestin-class promoter (~1.8 kb) + WPRE3 + polyA in a single AAV.

Method

  1. Parse three CIFs from existing AF3 results:
    • job2-mini-complex.cif (STRC 594-1775 + TMEM145) — REFERENCE, interface already mapped
    • job-f-shorter-mini-strc-700.cif (STRC 700-1775 solo) — CLINICAL candidate
    • job-h-strc-cterm-only.cif (STRC 1075-1775 solo) — AGGRESSIVE candidate
  2. Extract Cα atoms for canonical residues 1603-1749. Upper bound is 1749, not 1770, because NetGPI 1.1 predicts the GPI-anchor omega site at S1749. Residues 1750-1775 form the GPI prosequence, proteolytically cleaved during GPI attachment in the ER (see STRC GPI-Anchor Analysis). AF3 models them as a free flexible tail, but they do not exist in the mature membrane-anchored protein, so their apparent structural drift is a modeling artifact.
  3. Rigid-body align each truncation onto the reference using the top-15 TMEM145 hot contact residues (closest atomic distances in Job 2 interface extraction: aa 1607, 1630, 1648, 1650, 1669-76, 1695-1704). This preserves the binding-pocket frame and exposes peripheral drift honestly.
  4. After alignment, measure per-residue Cα deviation across the full bio-relevant interface zone (1603-1749).
  5. Report: hot-contact RMSD, full-zone RMSD, median/P75/P95/max per-residue deviation, fraction of residues within 3 Å drift.

Script: ~/STRC/models/mini_strc_interface_preservation.py. Results: ~/STRC/models/mini_strc_interface_preservation.json.

Result

ConstructRangeSize (aa)Hot-contact RMSDFull-zone RMSDMedian drift% residues < 3 ÅVerdict
Reference (job 2)594-17751182————interface at aa 1603-1749
Clinical700-177510760.17 Å0.70 Å0.19 Å98.0%preserved
Ultra-Mini1075-17757010.18 Å0.61 Å0.19 Å99.3%preserved (better!)

Both truncations preserve the TMEM145 binding interface to sub-Ångström accuracy at the median residue. Ultra-Mini (1075-1775) is structurally equivalent to the clinical construct in the binding zone — 99.3% of interface residues are within 3 Å Cα drift from the reference.

Peripheral outliers are at the GPI boundary

The top-5 outliers in both constructs are at canonical aa 1743-1749 (immediately N-terminal of the GPI omega site). This is the pre-GPI linker region, inherently flexible. Max deviation ~5 Å is consistent with the pre-GPI linker’s rotational freedom rather than a binding-interface disruption.

Interpretation

Both 700-1775 and 1075-1775 truncations preserve the TMEM145 anchoring interface.

Removing the LRR domain (approximately aa 700-1075) has zero measurable structural cost at the binding pocket. This is consistent with:

Engineering implications of Ultra-Mini (1075-1775)

MetricClinical 700-1775Ultra-Mini 1075-1775Delta
CDS length3,228 bp2,103 bp-1,125 bp
AAV headroom (ITR to ITR)1,472 bp2,597 bp+1,125 bp
Protein MW (pre-GPI)~120 kDa~78 kDa-42 kDa
CpG sites (pre-depletion)~156~95 (est)-40%
Glycosylation sites retained5/143/14-2

The 2,597 bp Ultra-Mini headroom unlocks:

  1. OHC-specific promoter — Prestin (Slc26a5) promoter is ~1.8 kb, standard CMV/CAG (~700 bp) fits both, but targeting specificity needs larger promoter.
  2. Full WPRE3 + bGH-polyA (regulatory cassette ~600 bp).
  3. Optional dual enhancer or miRNA binding sites for off-target silencing.
  4. Lower innate immunity via reduced CpG count and smaller cassette.

Caveats

  1. Structure prediction, not wet-lab. AF3 superposition shows fold preservation; does not prove functional equivalence. Wet-lab HEK coIP of Ultra-Mini × TMEM145 is the definitive test.
  2. AF3 cannot see membrane/glycosylation context. The Derstroff lesson applies. Glycosylation sites lost in Ultra-Mini (9 of 14) could affect trafficking or stability even if binding is preserved.
  3. 0.19 Å median drift is suspiciously low. AF3 may have converged to a canonical ARM fold across all truncations from training-data bias. The sub-Å result should be treated as a strong positive signal, not as definitive proof of structural identity in vivo.
  4. LRR domain function unknown. We assume LRR (aa ~700-1075) is a spacer. If LRR is involved in homodimerization (2026-04-17-liang-pcdh15-cryo-em-tip-link PCDH15 precedent), Ultra-Mini loses self-assembly capacity.
  5. Glycosylation site reduction. Ultra-Mini loses ~2 N-glycosylation sites vs clinical. May affect secretion efficiency even with IgK-SP.

Next computational steps

  1. AF3-Multimer: STRC 1075-1775 × TMEM145 full — direct structural test. Predict ipTM > 0.4 (matches Job 2) and contact residues 1603-1749. Submit as new AF3 job (af3_jobs_2026-04-20/strc_ultramini_x_tmem145.json). ✅ Resolved 2026-04-21 — ipTM 0.43 across 5 models, 23/41 contacts in GOLD zone, four of six canonical ARM-repeat clusters reproduced. Matches precedent, no regression. See STRC Ultra-Mini Full-Length TMEM145 AF3.
  2. AF3-Multimer: STRC 1075-1775 homodimer — if LRR domain is required for dimerization, Ultra-Mini solo vs dimer ipTM would split. Motivated by 2026-04-17-liang-pcdh15-cryo-em-tip-link PCDH15 precedent. ✅ Resolved 2026-04-21 — ipTM 0.28-0.30 with 94% C2 symmetry, self-contacts at aa 1579-1581 in ARM deep zone (inside Ultra-Mini zone). See STRC Homodimer Interface From CIF.
  3. CpG re-depletion for Ultra-Mini CDS — recompute CpG count at aa 1075-1775, regenerate CpG-free CDS (see STRC CpG Depletion Mini-STRC). ✅ Resolved 2026-04-21 — 0 CpG in payload at 3.65% CAI cost. See STRC Ultra-Mini CpG Depletion.
  4. OHC promoter shortlist — Prestin, Myo15, Pou4f3, Lhx3. Model expression level × specificity tradeoff. ✅ Resolved 2026-04-21 — B8 enhancer + WPRE3-compact winner. See STRC Ultra-Mini Promoter Shortlist.

All Ultra-Mini computational gates now passed. Next gate is wet-lab (order gBlock, clone pAAV construct, HEK coIP).

Ranking delta

  • STRC Mini-STRC Single-Vector Hypothesis: S-tier, no change to tier but evidence depth +1 and delivery score upgrade path opened. Prior delivery = 4; if Ultra-Mini (1075-1775) + Prestin promoter succeeds in AF3 validation, delivery → 5. Rationale: sub-Å structural equivalence at TMEM145 binding pocket validates the C-term-only construct, which doubles AAV headroom and unlocks OHC-specific expression cassette.
  • STRC Hypothesis Ranking: Mini-STRC “Next step” column updated from “complete AF3 ΔE1 × TMEM145 job suite” → “submit AF3 Ultra-Mini × TMEM145; if ipTM > 0.4, promote Ultra-Mini to clinical candidate”.
  • STRC mRNA-LNP Strategy B Full-Length: no change. Parallel track; this proof does not affect mRNA hypothesis. Still S-tier, delivery-gated.
  • STRC Pharmacochaperone Virtual Screen E1659A: no change. Maternal-allele fix; this proof is paternal-null therapy. Combo stack still valid.
  • STRC Piezoelectric TM Bioelectronic Amplifier: no change. Moonshot parallel track.
  • All other hypotheses: no change (this proof addresses Mini-STRC construct geometry only).

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