Mapping the interaction of cofilin with subdomain 2 on actin

Sabrina A. Benchaar, Yongming Xie, Martin Phillips, Rachel R.Ogorzalek Loo, Vitold E. Galkin, Albina Orlova, Mario Thevis, Andras Muhlrad, Steven C. Almo, Joseph A. Loo, Edward H. Egelman, Emil Reisler

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Abstract

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal α-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.

Original languageEnglish (US)
Pages (from-to)225-233
Number of pages9
JournalBiochemistry
Volume46
Issue number1
DOIs
StatePublished - Jan 9 2007

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Benchaar, S. A., Xie, Y., Phillips, M., Loo, R. R. O., Galkin, V. E., Orlova, A., Thevis, M., Muhlrad, A., Almo, S. C., Loo, J. A., Egelman, E. H., & Reisler, E. (2007). Mapping the interaction of cofilin with subdomain 2 on actin. Biochemistry, 46(1), 225-233. https://doi.org/10.1021/bi0610754