Identification of functional residues on Caenorhabditis elegans actin-interacting protein 1 (UNC-78) for disassembly of actin depolymerizing factor/cofilin-bound actin filaments.

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 beta-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.

Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting.

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.