Identification of Wiskott-Aldrich syndrome protein (WASP) binding sites on the branched actin filament nucleator Arp2/3 complex.

Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE

Original Publication: Washington, DC : National Academy of Sciences

Protein Conformation*; Actin Cytoskeleton/*metabolism ; Actin-Related Protein 2-3 Complex/*metabolism ; Saccharomyces cerevisiae/*metabolism ; Saccharomyces cerevisiae Proteins/*metabolism ; Wiskott-Aldrich Syndrome Protein/*metabolism; Actin Cytoskeleton/chemistry ; Actin-Related Protein 2-3 Complex/chemistry ; Amino Acid Sequence ; Binding Sites ; Protein Binding ; Protein Interaction Mapping ; Saccharomyces cerevisiae/growth & development ; Saccharomyces cerevisiae Proteins/chemistry ; Sequence Homology ; Wiskott-Aldrich Syndrome Protein/chemistry

Arp2/3 complex nucleates branched actin filaments important for cellular motility and endocytosis. WASP family proteins are Arp2/3 complex activators that play multiple roles in branching nucleation, but little is known about the structural bases of these WASP functions, owing to an incomplete understanding of how WASP binds Arp2/3 complex. Recent data show WASP binds two sites, and biochemical and structural studies led to models in which the WASP C segment engages the barbed ends of the Arp3 and Arp2 subunits while the WASP A segment binds the back side of the complex on Arp3. However, electron microscopy reconstructions showed density for WASP inconsistent with these models on the opposite (front) side of Arp2/3 complex. Here we use chemical cross-linking and mass spectrometry (XL-MS) along with computational docking and structure-based mutational analysis to map the two WASP binding sites on the complex. Our data corroborate the barbed end and back side binding models and show one WASP binding site on Arp3, on the back side of the complex, and a second site on the bottom of the complex, spanning Arp2 and ARPC1. The XL-MS-identified cross-links rule out the front side binding model and show that the A segment of WASP binds along the bottom side of the ARPC1 subunit, instead of at the Arp2/ARPC1 interface, as suggested by FRET experiments. The identified binding sites support the Arp3 tail release model to explain WASP-mediated activating conformational changes in Arp2/3 complex and provide insight into the roles of WASP in branching nucleation.
Competing Interests: The authors declare no conflict of interest.

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P41 GM103533 United States GM NIGMS NIH HHS; R01 GM092917 United States GM NIGMS NIH HHS

Keywords: Arp2/3 complex; actin; cross-linking; wasp

0 (Actin-Related Protein 2-3 Complex)
0 (LAS17 protein, S cerevisiae)
0 (Saccharomyces cerevisiae Proteins)
0 (Wiskott-Aldrich Syndrome Protein)

Date Created: 20180202 Date Completed: 20180801 Latest Revision: 20181113

20231215

PMC5816175

10.1073/pnas.1716622115

29386393