Mapping the Interaction of Cofilin with Subdomain 2 on Actin
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- 作者：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 ; $ ; Edw
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：January 9, 2007
- 年：2007
- 卷：46
- 期：1
- 页码：225 - 233
- 全文大小：313K
- 年卷期：v.46,no.1(January 9, 2007)
- ISSN：1520-4995
文摘
Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is akey regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs thefilaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactionswith subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a crypticcofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, whichtargets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal 
-actinand WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but wasstrongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysisof the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 onrecombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analyticalultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealedoligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bounda second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Underpolymerizing conditions the cross-linked AC formed mostly short filaments, which according to imagereconstruction 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 actinmutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actinto Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involvedin a weak binding of cofilin to G-actin.
-actinand WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but wasstrongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysisof the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 onrecombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analyticalultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealedoligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bounda second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Underpolymerizing conditions the cross-linked AC formed mostly short filaments, which according to imagereconstruction 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 actinmutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actinto Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involvedin a weak binding of cofilin to G-actin.