title: Structure of the formin-interaction domain of the actin nucleation-promoting factor Bud6.
abstract: Formin proteins and their associated factors cooperate to assemble unbranched actin filaments in diverse cellular structures. The Saccharomyces cerevisiae formin Bni1 and its associated nucleation-promoting factor (NPF) Bud6 generate actin cables and mediate polarized cell growth. Bud6 binds to both the tail of the formin and G-actin, thereby recruiting monomeric actin to the formin to create a nucleation seed. Here, we structurally and functionally dissect the nucleation-promoting C-terminal region of Bud6 into a Bni1-binding "core" domain and a G-actin binding "flank" domain. The ∼2-Å resolution crystal structure of the Bud6 core domain reveals an elongated dimeric rod with a unique fold resembling a triple-helical coiled-coil. Binding and actin-assembly assays show that conserved residues on the surface of this domain mediate binding to Bni1 and are required for NPF activity. We find that the Bni1 dimer binds two Bud6 dimers and that the Bud6 flank binds a single G-actin molecule. These findings suggest a model in which a Bni1/Bud6 complex with a 2:4 subunit stoichiometry assembles a nucleation seed with Bud6 coordinating up to four actin subunits.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Daqi
authors.initials: D
authors.lastname: Tu
authors.forename: Brian R
authors.initials: BR
authors.lastname: Graziano
authors.forename: Eunyoung
authors.initials: E
authors.lastname: Park
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Li
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Goode
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Eck
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008953:Models, Anatomic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
chemical_list: D000199:Actins; C107727:BUD6 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; D008840:Microfilament Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins