Mapping of Drebrin Binding Site on F-Actin |
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Authors: | Elena E Grintsevich Albina Orlova A Jimmy Ytterberg Mouna M Mikati Joseph A Loo Edward H Egelman |
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Institution: | 1 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA 2 Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA 3 Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA 4 Department of Biological Chemistry, University of California, Los Angeles, CA 90095, USA |
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Abstract: | Drebrin is a filament-binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full-length protein. Site-directed mutagenesis, electron microscopic reconstruction, and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and MTS1 1,1-methanediyl bis(methanethiosulfonate)]. Site-directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1 and that native cysteine 308 of drebrin is near C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, can link the N-terminal G-S extension of the recombinant DrABD to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different lengths (5.4-19 Å). These results suggest that the “core” DrABD is centered on actin subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of electron microscopic reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin-binding proteins. |
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Keywords: | ABP actin-binding protein mAbp1 mammalian actin-binding protein 1 DrABD actin-binding domain of drebrin (sequence 233-317) DrABD300 sequence 233-300 of drebrin drADF ADF-homology domain of drebrin (sequence 1-134) EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EM electron microscopic GST glutathione S-transferase MS mass spectrometry MS/MS tandem mass spectrometry MTS bis(methanethiosulfonate) MTS1 1 1-methanediyl bis(methanethiosulfonate) MTS8 3 6-dioxaoctane-1 8-diyl bis(methanethiosulfonate) MTS17 3 6 9 12 15-pentaoxaheptadecane-1 17-diyl bis(methanethiosulfonate) SD1-SD4 subdomains 1-4 WT wild type |
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