What they found
Crystal structures of WH2 domains from WASP, WAVE2, and WIP in complex with G-actin (ternary complex with DNase I as crystallization cofactor). Parallel ITC biochemistry comparing WH2 vs thymosin-β (Tβ) binding to actin. First structural evidence that short WH2 domains (~17 aa) can coexist with intersubunit contacts in F-actin; the canonical G-actin binding site (barbed-end groove between subdomains 1 and 3) is partly accessible on the filament surface.
Numbers that matter
WH2 G-actin binding affinities — ITC, 25°C, G-buffer (2 mM Tris pH 7.5, 0.2 mM CaCl₂, 0.2 mM ATP) — VALUES FROM FIGURE 1C (2026-04-25 update):
Figure 1C bar graph values read directly from main-text figure (MinerU image parse, 2026-04-25). SI Table 2 still inaccessible (PMC proof-of-work challenge blocks curl/fetch; PNAS 403); Fig. 1C displays the same ITC dataset graphically with error bars.
| Construct | Kd (µM) | Kd (nM) | Notes |
|---|---|---|---|
| Tβ4 (2-44) | 0.76 ± 0.12 | 760 ± 120 | Full-length Tβ4, reference |
| Tβ4 (2-33) | 3.10 ± 0.94 | 3100 ± 940 | N-terminal half only |
| Tβ4 (18-44) | no binding | — | C-terminal half only |
| Ciboulot (10-43) | 21.55 ± 3.80 | 21,550 | Tβ-family, weak |
| Ciboulot (54-82) | 53.48 ± 9.18 | 53,480 | Tβ-family, very weak |
| Ciboulot (87-119) | 6.25 ± 0.11 | 6,250 | Tβ-family |
| MIM (724-755) | 0.23 ± 0.04 | 230 ± 40 | Long WH2, strong |
| WIP (29-60) | 0.16 ± 0.01 | 160 ± 10 | Long WH2, strongest |
| WIP (29-46) | 2.15 ± 0.46 | 2,150 | N-terminal half |
| WIP (46-63) | >100 | >100,000 | C-terminal half, negligible |
| WASP (430-458) | 0.25 ± 0.03 | 250 ± 30 | Short WH2, weakest full-domain |
| WAVE2 (433-464) | 0.052 ± 0.003 | 52 ± 3 | Short WH2, strongest (Arg-Arg) |
| WAVE2 (450-464) | 26.53 ± 13.42 | 26,530 | C-terminal half, confirms N-helix drives binding |
Bold = constructs whose crystal structures are in this paper; these are the therapeutically relevant WH2 comparators.
Key ratios (cross-check with text):
- WAVE2/WASP = 0.25/0.052 = 4.8× (text: “~5-fold” — consistent)
- Tβ4/WASP = 0.76/0.25 = 3.0× (text says “~10-fold higher” — text likely refers to WAVE2/Tβ4 = 14.6× or to average WH2 family vs Tβ4 average including ciboulot)
CRITICAL NOTE on model value: WH2_KD_GACTIN_M = 200 nM is between WIP (160 nM) and WASP (250 nM) — a conservative mid-range for de novo WH2 constructs of unknown sequence type. WAVE2 (52 nM) is the favorable outlier due to Arg-Arg in LKKT forming salt bridges with Asp-24/Asp-25 on actin.
SI Table 2 retrieval history:
- Anna’s Archive (DOI, title+supplementary, MD5 searches): SI not separately indexed — 4 attempts, 0 hits
- PMC (pnas_0507021102_2.pdf): proof-of-work challenge blocks non-browser access
- PNAS publisher: 403 on all SI URL patterns
- Resolution: Fig. 1C in main text is the same ITC dataset — values read directly from bar graph image. Status upgraded ⚠ → ✅.
WH2 × F-actin side-binding — NOT MEASURED in this paper:
- The paper shows WH2 binding site (barbed-end groove, subdomains 1/3) is ALSO the site of longitudinal actin–actin contacts in the filament long-pitch helix
- Short WH2 domains (~17 aa) can “coexist with intersubunit contacts in F-actin” structurally, but this means nucleation (WH2 templating monomers along a strand), not side-binding of preformed filaments
- No co-sedimentation or F-actin binding Kd measured
Tβ4 × F-actin: weak cooperative binding, Kd = 5–10 mM (cited from Husson 2010) This is 3 orders of magnitude weaker than G-actin binding — the C-terminal α-helix in Tβ4 interferes with filament contacts, but the same region being absent in WH2 doesn’t mean WH2 side-binds better; it just means WH2 doesn’t lock monomer as completely.
Why WH2 × F-actin side-binding is structurally disfavored
The N-terminal amphipathic helix of WH2 binds in the cleft between actin subdomains 1 and 3. In the filament (Holmes model), this same cleft is occupied by longitudinal actin–actin contacts along the long-pitch helix — it is partially buried. Residual surface accessibility does not constitute a high-affinity independent binding site. No evidence that WH2 can bind to the side of a preformed filament with affinity < 1 mM. The model’s WH2_KD_FACTIN_M = 5 μM is optimistic; the real value is likely 1–100 mM or unmeasurable.
Connections
[source]STRC H09 WH2 F-actin Bundling Hypothesis — provides structural rationale and Kd reference range for G-actin binding; establishes F-actin side-binding as unmeasured and structurally disfavored- Hydrogel Phase 4d F-actin Bundling Model — WH2_KD_GACTIN_M defensible at 50–200 nM for favorable WH2 constructs; WH2_KD_FACTIN_M = 5 μM is speculative/optimistic
[see-also]Recipe — Short WH2 Actin Binder Design — distilled design rules (P1)[see-also]Short vs Long WH2 Domain Classification — structural sub-family split (P2)[see-also]Tandem WH2 Filament Nucleation Mechanism — nucleation geometry (P2)[see-also]actin-kinetics — parameter table consumer[applies]h09 hub