title: High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1.
abstract: The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex.
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Joern
authors.initials: J
authors.lastname: Linkner
authors.forename: Gregor
authors.initials: G
authors.lastname: Witte
authors.forename: Theresia
authors.initials: T
authors.lastname: Stradal
authors.forename: Ute
authors.initials: U
authors.lastname: Curth
authors.forename: Jan
authors.initials: J
authors.lastname: Faix
mesh_terms: D015153:Blotting, Western; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray / Q000379:methods*; D004023:Dictyostelium / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D014461:Ultracentrifugation; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; C491975:SCAR protein, Dictyostelium; D051300:Wiskott-Aldrich Syndrome Protein Family