Cofilin is an actin regulatory protein that binds to both monomeric and filamentous actin, and has filament severing activity. Although crystal structures for the monomeric forms of both G-actin and cofilin have been described, the structure of the binary cofilin-G-actin complex is not available. Synchrotron protein footprinting is used to identify specific side chain residues on the cofilin surface that are buried in the formation of the cofilin-G-actin binary complex. Exposure to synchrotron X-rays results in stable oxidative modifications of aromatic, aliphatic, and sulfur-containing side chains, with the rate of modification for a particular residue being dependent on its intrinsic reactivity and solvent accessibility. The rates of modification were monitored for a number of peptides generated by digestion of oxidized cofilin, both in isolation and in its binary complex with G-actin. After binding to G-actin takes place, a significant decrease in modification rates, indicating protection of side chain groups, is seen for cofilin peptides corresponding to residues 4-20, 10-17, 83-96, 91-105, and 106-117. A number of other peptides show no change in reactivity, and are presumed to represent regions distal to the binding site. Tandem mass spectrometry demonstrates that residues Leu 13, Pro 94, Met 99, and Leu 108 and 112 directly participate in the binding interface. These results are generally consistent with, and complementary to, the results of previous site-directed mutagenesis studies and extend our understanding of the G-actin binding surface of cofilin.
Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin. AIP1 also caps the barbed end of ADF/cofilin-bound actin filament. However, the mechanism by which AIP1 interacts with ADF/cofilin and actin is not clearly understood. We determined the crystal structure of Caenorhabditis elegans AIP1 (UNC-78), which revealed 14 WD40 modules arranged in two seven-bladed -propeller domains. The structure allowed for the mapping of conserved surface residues, and mutagenesis studies identified five residues that affected the ADF/cofilin-dependent actin filament disassembly activity. Mutations of these residues, which reside in blades 3 and 4 in the N-terminal propeller domain, had significant effects on the disassembly activity but did not alter the barbed end capping activity. These data support a model in which this conserved surface of AIP1 plays a direct role in enhancing fragmentation/depolymerization of ADF/cofilin-bound actin filaments but not in barbed end capping.Actin filaments are the core components of the highly dynamic actin cytoskeleton, which plays an essential role in a wide range of biological processes. Regulated fragmentation and depolymerization of actin filaments are important for disassembling specific cytoskeletal structures and maintaining high concentrations of monomeric actin, thereby enhancing actin filament turnover. Actin-depolymerizing factor (ADF) 1 / cofilin plays a central role in the disassembly process by accelerating monomer dissociation from the pointed ends and severing filaments (reviewed in Refs. 1-4).Actin-interacting protein 1 (AIP1) is a unique regulator of ADF/cofilin-mediated actin dynamics (reviewed in Ref. 5). The interaction between AIP1 and actin was originally detected by a yeast two-hybrid system (6). In vitro, AIP1 alone has negligible or very weak effects on actin dynamics, whereas, in the presence of ADF/cofilin, AIP1 enhances filament fragmentation (7-9). In addition, AIP1 has the ability to cap barbed ends and bind to the side of ADF/cofilin-bound filaments (10). Although the capping activity has been suggested to enhance filament fragmentation by preventing reannealing of the severed filaments (10, 11), direct microscopic observation of the effects of AIP1 on ADF/cofilin-bound filaments indicates that AIP1 actively disassembles ADF/cofilin-bound filaments, and simple barbed end capping by other capping agents does not enhance disassembly by ADF/cofilin (12). At present, the biological significance of the capping activity of AIP1 is not well understood.The mechanism by which AIP1 selectively interacts with ADF/cofilin-bound actin filaments is unknown. Actin residues in subdomains 3 and 4 participate in the two-hybrid interaction with yeast AIP1 (6). The AIP1-interacting residues in subdomain 3 are also required for interaction with cofilin (9), suggesting that AIP1 directly interacts with subdomain 4 of actin and indirectly with subdomain 3, which might be mediate...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.