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. 2015 Aug 4;23(8):1492-1499.
doi: 10.1016/j.str.2015.05.015. Epub 2015 Jun 25.

Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif

Eunyoung Park  1 , Brian R Graziano  2 , Wei Zheng  1 , Mikael Garabedian  2 , Bruce L Goode  2 , Michael J Eck  3
Affiliations

Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif

Eunyoung Park et al. Structure. .

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.

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Figures

Figure 1
Figure 1. Crystal structure of Bud6flank in complex with actin
A, Domain structures of Bud6 and the formin Bni1. B, Overview of the complex. Actin is shown in a surface representation, and Bud6 as a yellow ribbon. ATP is present in the nucleotide-binding cleft of actin. C and D, Stereo views of the interactions of helices αA and αB, respectively. Actin is shown as a blue ribbon, Bud6 as a yellow ribbon. Selected side chains are shown in stick form, Bud6 residues are labeled in bold text and actin residues in plain text. Electron density in the region of helix αA is shown in Figure S1. E, Evolutionary conservation of Bud6flank. Selected Bud6 sequences are aligned and shaded according to rate of evolutionary variation based on analysis of sequences of Bud6 from 46 fungal species as previously described (Tu et al., 2012). Analysis was carried out with the CONSURF server (Ashkenazy et al., 2010); shading ranges from dark magenta to teal (most conserved to most variable, respectively). Secondary structure elements are indicated above the alignment, and residues in contact with actin (as determined by a 4.0 Å distance cutoff) are indicated by grey dots.
Figure 2
Figure 2. Comparison of the Bud6 actin-binding domain with WH2 domains
A, Superposition of actin-bound Bud6flank (yellow) and the WH2 domains of WIP (WASP-interacting protein, dark blue, PDB ID 2A41) and Spire (purple, PDB ID 3MN7). Structures were superimposed using the actin-binding portion of each of the three structures; the light blue surface corresponds to the actin-binding portion of the present structure. The WH2 domain helix overlaps closely with helix αA of Bud6flank, but there is no equivalent of helix αB in the WH2 domains. Both Bud6flank and these two WH2 domains position an arginine residue between Glu167 and Tyr169 in actin (inset). Note also that the Bud6flank helix is one turn longer than that of the WH2 domains. See also Figure S2. B, Comparison of Bud6flank and WH2 domain sequences. Sequences of Bud6flank and selected WH2 domains are shown, with conserved actin-binding residues boxed. Respective secondary structures are shown above the sequences, and the structurally overlapping region of Bud6flank and the WH2 motif is indicated. Note that there is no equivalent of the “LKKT” WH2 sequence motif in Bud6flank.
Figure 3
Figure 3. Structure-function analysis of the Bud6-actin interaction
A, Concentration-dependent effects of wild type and mutant C-Bud6 polypeptides. 2 μM monomeric actin was polymerized in the presence of 10 nM Bni1 FH1-FH2-C and indicated concentrations of wild type or mutant C-Bud6 (550-788). Fold increase in actin assembly activity (relative to Bni1 FH1-FH2-C alone) is plotted as a function of C-Bud6 concentration. B, As in A, but the activity of intact C-Bud6 (residues 550-788) is compared with C-Bud6Δ740-788 (residues 550-739). See Figure S3 for raw actin assembly curves.
Figure 4
Figure 4. Mechanistic implications of the Bud6 structure and mode of actin binding
A, The Bud6flank binding site on actin overlaps with that of other actin-assembly factors and is partially blocked in F-actin. Crystal structures of Bud6flank, profilin, and the Bni1 FH2 domain in complex with actin are shown in the same orientation. The barbed-end groove occupied by helix αA in Bud6 is also part of the binding surface for profilin and the FH2 domain. The groove is also blocked in F-actin by the DNAse I binding loop (arrow) of a longitudinally apposed subunit (medium blue) in the helical filament. Three actin subunits of a filament are drawn based on the X-ray fiber diffraction structure of F-actin (PDB ID 2ZWH); examination of a cryo-electron microscopy reconstruction leads to the same conclusion (PDB ID 3MFP, not shown). B, Schematic summary of available structural information for the Bni1 FH2 domain and C-Bud6 and their interactions with actin. Components are drawn approximately to scale, and the illustration is based on structures of the Bni1 FH2 domain (green) bound to actin (blue), the Bud6core domain (yellow and red), and Bud6flank in complex with actin (yellow or red, with actin in gray). No structure is available for Bud6core in complex with Bni1, but biochemical studies map the binding interaction to the “tail” of Bni1, which lies just C-terminal to the long “αT” helix of the FH2 domain. The Bni1 tail and the ~20 residue linker that connects Bud6 flank and core domains are shown as dotted lines, because they are not present in available crystal structures. The Bni1 FH2 dimer is thought to promote nucleation by bridging between two or more actin subunits in a filament-like orientation, via contacts of its “knob” and “post” elements. C, Superposition of Bud6flank on a Bni1/actin complex. Three actin subunits and a Bni1 FH2 domain dimer from the crystal structure of the complex (PDB ID 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). Bud6flank (yellow) is docked based on superposition of the present structure with the light-blue actin subunit. The Bud6 binding site on the medium and dark blue actin subunits is blocked by contact with the FH2 domain, but it is accessible on the light blue subunit, which is in contact with only the post-site of the FH2 domain. We speculate that this mode of interaction could allow Bud6 to contribute to filament nucleation by the FH2 domain (see text). There is a modest steric clash between the end of Helix αB in Bud6 and the opposite subunit in the FH2 dimer (rotated view), but the precise orientation of the two FH2 subunits that leads to this clash arises from crystallographic symmetry and is not thought to be directly relevant to Bni1-mediated nucleation. Note that one of the flexible linkers connecting the two halves of the FH2 dimer is not illustrated; due to an artifact in the crystal structure, it connects to an adjacent FH2 subunit in the lattice rather than closing the FH2 dimer.
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