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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Actin Depolymerizing Factors
Actins
Yeast
Cofilin 1
Yeasts
Actin Cytoskeleton
Sedimentation
Cofilin 2
Electron microscopy
Electron Microscopy
Destrin

ASJC Scopus subject areas

  • Biochemistry

Cite this

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

Mapping the interaction of cofilin with subdomain 2 on actin. / Benchaar, Sabrina A.; Xie, Yongming; Phillips, Martin; Loo, Rachel R Ogorzalek; Galkin, Vitold E.; Orlova, Albina; Thevis, Mario; Muhlrad, Andras; Almo, Steven C.; Loo, Joseph A.; Egelman, Edward H.; Reisler, Emil.

In: Biochemistry, Vol. 46, No. 1, 09.01.2007, p. 225-233.

Research output: Contribution to journalArticle

Benchaar, SA, Xie, Y, Phillips, M, Loo, RRO, Galkin, VE, Orlova, A, Thevis, M, Muhlrad, A, Almo, SC, Loo, JA, Egelman, EH & Reisler, E 2007, 'Mapping the interaction of cofilin with subdomain 2 on actin', Biochemistry, vol. 46, no. 1, pp. 225-233. https://doi.org/10.1021/bi0610754
Benchaar SA, Xie Y, Phillips M, Loo RRO, Galkin VE, Orlova A et al. Mapping the interaction of cofilin with subdomain 2 on actin. Biochemistry. 2007 Jan 9;46(1):225-233. https://doi.org/10.1021/bi0610754
Benchaar, Sabrina A. ; Xie, Yongming ; Phillips, Martin ; Loo, Rachel R Ogorzalek ; Galkin, Vitold E. ; Orlova, Albina ; Thevis, Mario ; Muhlrad, Andras ; Almo, Steven C. ; Loo, Joseph A. ; Egelman, Edward H. ; Reisler, Emil. / Mapping the interaction of cofilin with subdomain 2 on actin. In: Biochemistry. 2007 ; Vol. 46, No. 1. pp. 225-233.
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AU - Galkin, Vitold E.

AU - Orlova, Albina

AU - Thevis, Mario

AU - Muhlrad, Andras

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