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Revealing general principles of actin nucleation through structural and mechanistic studies of bacterial effector VopL from Vibrio Parahaemolyticus

Jacob Zahm Research Image

VopL is a type-III secreted effector protein from Vibrio parahaemolyticus that induces stress fiber formation in cells and exhibits potent actin nucleation activity in vitro.

The molecule possesses two distinct regions:

  • An N-terminal sequence that contains three tandem actin-binding WH2 domains
  • A unique VopL C-terminal domain (VCD)

Our lab showed that the N-terminal sequence, in the absence of the VCD, lacks nucleation activity, and that the VCD, in the absence of the N-terminal sequences, exhibits modest nucleation activity; inclusion of both sequences results in a synergistic enhancement of potency. To understand the mechanism of VCD-mediated nucleation, I determined the crystal structure of the VCD in complex with actin. The structure reveals that the VCD operates by organizing actin monomers into a near-filamentous actin nucleus that likely serves as a template for addition of subsequent monomers.

While similar to an actin filament, the arrangement of actin monomers in the VCD-induced nucleus deviates from ideality. We posit that these deviations give rise to strain, relief of which drives VopL disengagement from the nascent filament. Such deviations may also drive the dynamic behavior of other nucleation factors in the formin family.

I am currently working to understand the relationship between strain and the dynamic behavior of actin nucleation factors. The WH2 domains of VopL recruit actin monomers and deliver them to the VCD, thus facilitating the formation of a nucleus. However, it is not known how, or at what stage of nucleation the WH2 domains dissociate from the nascent filament.

A mechanistic understanding of WH2-mediated actin delivery and subsequent dissociation in VopL will likely aide in our understanding of other nucleation systems that rely on WH2-domains, such as the Arp2/3 complex, and other tandem WH2 nucleators such as Spire and Cordon-bleu.

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