title: Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry.
abstract: Actin-related protein (Arp) 2/3 complex nucleates new branches in actin filaments playing a key role in controlling eukaryotic cell motility. This process is tightly regulated by activating factors: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We used radiolytic protein footprinting with mass spectrometry in solution to probe the effects of nucleotide- and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting approaches. First, Arp2/3 is the most complex macromolecular assembly yet examined; second, only a few picomoles of Arp2/3 was required for individual experiments. In terms of structural biology of Arp 2/3, we find that ATP binding induces conformational changes within Arp2/3 complex in Arp3 (localized in peptide segments 5-18, 212-225, and 318-327) and Arp2 (within peptide segment 300-316). These data are consistent with nucleotide docking within the nucleotide clefts of the actin-related proteins promoting closure of the cleft of the Arp3 subunit. However, ATP binding does not induce conformational changes in the other Arp subunits. Arp2/3 complex binds to WASp within the C subdomain at residue Met 474 and within the A subdomain to Trp 500. Our data suggest a bivalent attachment of WASp to Arp3 (within peptides 162-191 and 318-329) and Arp2 (within peptides 66-80 and 87-97). WASp-dependent protections from oxidation within peptides 54-65 and 80-91 of Arp3 and in peptides 300-316 of Arp2 suggest domain rearrangements of Arp2 and Arp3 resulting in a closed conformational state consistent with an "actin-dimer" model for the active state.
authors.affiliation: Case Center for Proteomics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
authors.forename: Janna G
authors.initials: JG
authors.lastname: Kiselar
authors.forename: Rachel
authors.initials: R
authors.lastname: Mahaffy
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pollard
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000737:chemistry* / Q000378:metabolism*; D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000495:Allosteric Site; D000818:Animals; D002417:Cattle; D013058:Mass Spectrometry / Q000379:methods*; D009711:Nucleotides / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D009711:Nucleotides; D051316:Wiskott-Aldrich Syndrome Protein; D014364:Tryptophan; D000255:Adenosine Triphosphate; D010100:Oxygen