Otomo T, Tomchick DR, Otomo C, Panchal SC, Machius M, Rosen MK.. Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain. Nature 433: 488-494

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Nature (Impact Factor: 41.46). 03/2005; 433(7025):488-94. DOI: 10.1038/nature03251
Source: PubMed


The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.

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Available from: Mischa Machius
    • "Three actin subunits and a Bni1 FH2 domain dimer from the crystal structure of the complex (PDB: 1Y64) are shown in shades of blue and green, respectively. The interaction with the formin arranges the actin subunits in a filament-like orientation that is proposed to lead to formation of a nascent filament (Otomo et al., 2005b). Bud6 flank (yellow) is docked based on superposition of the present structure with the light-blue actin subunit. "
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    ABSTRACT: In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. Copyright © 2015 Elsevier Ltd. All rights reserved.
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    • "We previously found that three mutations in Capu's post region (K851A, K853A, and K858A) similarly enhance Capu's actin assembly activity in bulk assays (Roth-Johnson et al., 2014). Otomo et al. (2005) also demonstrated that two knob mutations in Bni1p slightly enhance its in vitro activity. Although rare, mutations that accelerate, rather than slow, a formin's actin assembly activity have now been identified in the loop, post, and knob regions of the FH2 domain. "
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    ABSTRACT: During Drosophila development, the formin actin nucleator Cappuccino (Capu) helps build a cytoplasmic actin mesh throughout the oocyte. Loss of Capu leads to female sterility presumably because polarity determinants fail to localize properly in the absence of the mesh. To gain deeper insight into how Capu builds this actin mesh, we systematically characterized seven capu alleles, which have missense mutations in Capu's formin homology 2 (FH2) domain. We report that all seven alleles have deleterious effects on fly fertility and the actin mesh in vivo, but have strikingly different effects on Capu's biochemical activity in vitro. Using a combination of bulk and single filament actin-assembly assays, we find that the alleles differentially affect Capu's ability to nucleate and processively elongate actin filaments. We also identify a unique "loop" in the lasso region of Capu's FH2 domain. Removing this loop enhances Capu's nucleation, elongation, and F-actin bundling activities in vitro. Together, our analysis of the loop and the seven missense mutations provides mechanistic insight into formin function in general and Capu's role in the Drosophila oocyte in particular. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Mar 2015 · Molecular biology of the cell
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    • "The majority of resides are involved in known actin–actin or actin–binding-partner contacts. Buried (B, blue) indicates buried residues in the G-actin structure (Wang et al., 2010), F-actin (F, mustard) indicates residues that are in the F-actin interfaces (von der Ecken et al., 2014), Arp2/3 (A, pink) (Robinson et al., 2001; Volkmann et al., 2001), Formins (F, green) (Otomo et al., 2005; Thompson et al., 2013), Myosin (M, orange) (Behrmann et al., 2012), Cofilin (T, cyan) (Paavilainen et al., 2008) and Profilin (P, red) (Schutt et al., 1993) indicate interacting residues with each protein. The twinfilin–actin structure is used here as a model for the cofilin–actin interactions. "
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    ABSTRACT: The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity.
    Full-text · Article · Mar 2015 · Journal of Cell Science
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