Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments
Abstract
We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET_(eff)) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ET_(eff) between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.
Additional Information
© 2018 National Academy of Sciences. Published under the PNAS license. Edited by Gary G. Borisy, The Forsyth Institute, Cambridge, MA, and approved August 2, 2018 (received for review October 6, 2017). Published ahead of print August 27, 2018. We thank Shih-Chieh Ti for providing the S. pombe strains used in the ensemble FRET experiments and his advice on the project and three expert reviewers and Roberto Dominquez, who each provided suggestions that substantially improved the paper. This work was supported by National Institute of General Medical Sciences of the National Institutes of Health Grants R01GM026338 (to T.D.P.) and R01NS079955 (to E.R.), an International Fellowship from the American Association of University Women (to S.E.-S.), American Heart Association Fellowship 13PRE16570013 (to L.A.M.), and the Yale Integrated Graduate Program in Physical and Engineering Biology (S.E.-S. and L.A.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author contributions: S.E.-S., L.A.M., E.R., and T.D.P. designed research; S.E.-S., L.A.M., and S.Z.C. performed research; S.E.-S., L.A.M., S.Z.C., E.R., and T.D.P. analyzed data; and S.E.-S., L.A.M., S.Z.C., E.R., and T.D.P. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717594115/-/DCSupplemental.Attached Files
Published - E8642.full.pdf
Supplemental Material - pnas.1717594115.sapp.pdf
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Additional details
- PMCID
- PMC6140485
- Eprint ID
- 89188
- Resolver ID
- CaltechAUTHORS:20180828-072940274
- R01GM026338
- NIH
- R01NS079955
- NIH
- American Association of University Women
- 13PRE16570013
- American Heart Association
- Yale University
- Created
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2018-08-28Created from EPrint's datestamp field
- Updated
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2022-03-07Created from EPrint's last_modified field