Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry

Janna G. Kiselar, Rachel Mahaffy, Thomas D. Pollard, Steven C. Almo, Mark R. Chance

Research output: Contribution to journalArticle

48 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1552-1557
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number5
DOIs
StatePublished - Jan 30 2007

Fingerprint

Actin-Related Protein 2-3 Complex
Mass Spectrometry
Adenosine Triphosphate
Peptides
Actins
Nucleotides
Actin-Related Protein 2
Actin-Related Protein 3
Protein Footprinting
Macromolecular Substances
Protein Subunits
Eukaryotic Cells
Actin Cytoskeleton
Cell Movement
Proteins

Keywords

  • Actin
  • Dynamics
  • Footprinting

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry. / Kiselar, Janna G.; Mahaffy, Rachel; Pollard, Thomas D.; Almo, Steven C.; Chance, Mark R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 5, 30.01.2007, p. 1552-1557.

Research output: Contribution to journalArticle

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