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| Title | NPF binding to Arp2 is allosterically linked to the release of ArpC5's N-terminal tail and conformational changes in Arp2/3 complex. |
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| Journal, issue, pages | Proc Natl Acad Sci U S A, Vol. 122, Issue 8, Page e2421557122, Year 2025 |
| Publish date | Feb 25, 2025 |
 Authors | Andrew J Saks / Kyle R Barrie / Grzegorz Rebowski / Roberto Dominguez /  |
| PubMed Abstract | Arp2/3 complex generates branched actin networks essential for numerous motile functions of the cell. It comprises seven subunits: actin-related proteins (Arps) 2 and 3 and five scaffolding subunits ...Arp2/3 complex generates branched actin networks essential for numerous motile functions of the cell. It comprises seven subunits: actin-related proteins (Arps) 2 and 3 and five scaffolding subunits (ArpC1-5). The complex adopts two major conformations: inactive, with the Arps interacting end-to-end, and active, with the Arps aligned side-by-side like subunits in the actin filament. Activation involves several cofactors, including ATP, WASP-family nucleation-promoting factors (NPFs), actin monomers, and the mother actin filament. NPFs bind to two sites, one on Arp2-ArpC1 and one on Arp3, delivering actin subunits at the barbed end of the Arps to initiate branch elongation. However, the mechanisms by which each NPF drives the equilibrium toward activation remain unclear. We present two cryo-electron microscopy (cryo-EM) structures of Arp2/3 complex at 2.9-Å resolution: one with NPFs bound to Arp3 and ArpC1 but not Arp2 and another with NPFs bound to Arp3 and Arp2-ArpC1. The structures reveal that NPF binding to Arp2 is allosterically linked to the release of ArpC5's N-terminal tail from Arp2 and conformational changes in Arp2, including closure of its ATP-binding cleft and partial rotation and translation toward its position in the active complex at the branch. Previous work identified another allosteric switch linking NPF binding to Arp3 with the release of its inhibitory C-terminal tail, which we also observe. In summary, both NPF-binding sites induce allosteric changes in Arp2/3 complex, collectively shifting the equilibrium toward activation. |
 External links | Proc Natl Acad Sci U S A / PubMed:40042350 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 2.85 - 3.41 Å |
| Structure data |  EMDB-46913: local refinement of Arp3 and ArpC2  EMDB-46924: local refinement of ArpC3 for Arp 2/3 complex bound to NPFs  EMDB-46925: local refinement of arpC5, arpC1 and arp2 for arp 2/3 complex with 1 of 2 NPFs dissociated and arpC5 tail bound  EMDB-46926: Local refinement of ArpC4 for Arp 2/3 complex bound to NPFs  EMDB-46934: local refinement of arpC5, arpC1 and arp2 for arp 2/3 complex 2 NPFs bound and arpC5 tail mostly dissociated EMDB-46992, PDB-9dlx: EMDB-46993, PDB-9dlz: |
| Chemicals |  ChemComp-ATP:  ChemComp-MG: |
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 Keywords | STRUCTURAL PROTEIN / actin / arp 2-3 complex / N-WASP / nucleation promoting factor |
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