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):
- Exact Kd values are in Table 2 (SI, not open-access). Narrative in text only gives relatives:
- WAVE WH2: strongest binder (5× higher affinity than WASP WH2)
- WASP WH2: weakest binder in series
- All WH2 domains: ~10× higher affinity than Tβ domains
- Since Tβ4 Kd = 1 μM (Husson 2010, Xue 2014), this implies WH2 Kd range ~50–200 nM
- WASP WA domain (WH2 + C region together): Kd = 0.6 μM from Rohatgi 2000 (cited)
- Isolated WASP WH2 (V domain alone, from Padrick/Kim 2011 citing this paper): Kd ≈ 3.1 μM
CRITICAL NOTE on “100 nM” in model: The ~10× boost vs Tβ4 is for construct-dependent WH2 variants. The isolated WASP WH2 V-domain alone is ~3 μM, not 100 nM. The 100 nM figure likely applies to long WH2 constructs (WIP-type) or in-context domains with favorable electrostatics (WAVE with Arg-Arg in LKKT gives extra salt bridges). Model parameter WH2_KD_GACTIN_M = 200 nM is near the favorable end; range is 50 nM–3 μM depending on construct.
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
- WH2 Actin Binding Mechanism — barbed-end groove binding