title: Filament assembly by Spire: key residues and concerted actin binding.
abstract: The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Rasson
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA.
authors.forename: Justin S
authors.initials: JS
authors.lastname: Bois
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA.
authors.forename: Duy Stephen L
authors.initials: DSL
authors.lastname: Pham
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA.
authors.forename: Haneul
authors.initials: H
authors.lastname: Yoo
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California Los Angeles, Paul D. Boyer Hall, 611 Charles E. Young Drive East, Box 951570, Los Angeles, CA 90095-1570, USA. Electronic address: margot@chem.ucla.edu.
authors.forename: Margot E
authors.initials: ME
authors.lastname: Quinlan
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000199:Actins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; C402385:spir protein, Drosophila
keywords: Spir; WH2; actin; cytoskeleton; nucleation