Recipe — Short WH2 Actin Binder Design
How to design a synthetic WH2-class peptide that binds G-actin with nanomolar affinity. Distilled from Chereau et al. 2005 PNAS (WASP, WAVE2, WIP crystal structures) [Chereau 2005, p.16644–16649]. Use when scaffolding a new actin-targeting therapeutic peptide for h09 hydrogel work or any WH2-anchored construct.
Minimum design rules
| # | Rule | Source detail |
|---|---|---|
| 1 | N-terminal amphipathic α-helix, three turns (~12 aa). | “three-turn amphiphilic α-helix, with its hydrophobic side embedded within the cleft formed between actin subdomains 1 and 3” [Chereau 2005, p.16645]. Two-turn (Tβ4-style) loses affinity. |
| 2 | Conserved N-cap Arg + C-cap Gly on the α-helix. | ”conserved Arg at the N terminus and a conserved Gly at the C terminus, both absent in Tβ proteins” [p.16645]. The Arg is the first residue contacting actin; the Gly shifts the linker forward relative to Tβ. |
| 3 | LKKT / LKKV motif 2–3 residues C-terminal to the helix. | ”conserved Leu in the LKKT motif and a second hydrophobic amino acid in the linker, two amino acids N-terminal to this Leu, bind together in a hydrophobic pocket in actin” [p.16645]. Final residue Thr/Val/Ala only — larger side chains incompatible. |
| 4 | Short linker (≤ 4 aa) between helix and LKKT. | Tβ linker is 3 aa longer and forms a helical turn — “constrains this important interaction into a less favorable conformation” [p.16645]. Short linker = ~10× tighter binding. |
| 5 | Optional affinity boost: replace LKKT Lys-Lys with Arg-Arg. | ”two Arg residues in the LKKT sequence of WAVE…form salt bridges with actin residues Asp-24 and Asp-25” [p.16645]. WAVE2 (LRRT) Kd = 52 nM vs WASP (LKKT) Kd = 250 nM — ~5× boost [Fig. 1C]. |
| 6 | Stop after LKKT for filament-compatible construct (~17 aa total). | Short WH2 (WASP/WAVE) “do not interfere with intersubunit contacts in F-actin” [p.16646–16647]. Required for tandem-WH2 nucleation lining up monomers along a filament strand. |
| 7 | Extend with β-strand + Ser if monomer-sequestration is goal (~27 aa, long WH2). | Long WH2 (WIP/MIM) adds residues that “form a β-strand that runs parallel to a β-sheet in actin subdomain 1” plus a Ser that “penetrates deeper into the nucleotide cleft” [p.16645]. Higher affinity (WIP 160 nM) but disrupts filament contacts. |
Affinity expectations (target window)
ITC-measured G-actin Kd at 25 °C, G-buffer (2 mM Tris pH 7.5, 0.2 mM CaCl₂, 0.2 mM ATP) [Chereau 2005, Fig. 1C]:
| Construct | Kd | Class |
|---|---|---|
| WAVE2 (433–464) | 52 ± 3 nM | short, Arg-Arg upgraded |
| WIP (29–60) | 160 ± 10 nM | long |
| MIM (724–755) | 230 ± 40 nM | long |
| WASP (430–458) | 250 ± 30 nM | short, baseline |
| Tβ4 (2–44) | 760 ± 120 nM | reference (not WH2) |
Defensible design target: 50–250 nM for a short WH2 with the Arg-Arg upgrade; 150–250 nM for plain LKKT. Below 50 nM is outside the measured WH2 envelope without tandem-copy avidity.
What this recipe does NOT design
- F-actin side-binding. The same subdomain 1/3 cleft is occupied by longitudinal actin–actin contacts in F-actin. Single-WH2 side-binding Kd is unmeasured in any paper; closest analog (Tβ4 × F-actin) is 5–10 mM. Avidity from multi-WH2 scaffolds (h09 RADA16 strategy) is the only path; expect Phase 2c wet-lab to gate this — see actin-kinetics and 2026-04-23-chereau-wh2-actin-pnas.
- Nucleotide exchange tuning. Inhibition is a function of the N-terminal helix in the cleft (locks actin near hinge) — comes “for free” with rule #1, not separately tunable.
- Profilin-handoff geometry. Requires N-terminal Pro-rich region upstream of the WH2; out of scope for this recipe.
Anti-patterns
- Two-turn α-helix (Tβ4-like) — drops affinity ~3×.
- Long linker between helix and LKKT — Tβ-like geometry, ~10× weaker.
- LKKT terminal residue larger than Thr/Val/Ala — pocket clash.
- Skipping the conserved Arg N-cap — first-contact residue.
Connections
[source]2026-04-23-chereau-wh2-actin-pnas[part-of]STRC Synthetic Peptide Hydrogel HTC[see-also]Short vs Long WH2 Domain Classification[see-also]Tandem WH2 Filament Nucleation Mechanism[see-also]actin-kinetics[applies]h09 hub