Structural Basis of Actin Filament Nucleation by Tandem W Domains

• The structure of a dimeric actin nucleus with Cobl, an actin nucleator, was solved
• Actin and Cobl form a stable nucleus in a configuration different from F-actin
• JMY, but not Spire, uses a similar mechanism to Cobl for actin nucleation
• The double-mutant strategy opens the way for mechanistic study of actin nucleation

SummarySpontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.

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