Identification of Functional Residues on Caenorhabditis elegans Actin-interacting Protein 1 (UNC-78) for Disassembly of Actin Depolymerizing Factor/Cofilin-bound Actin Filaments

Kurato Mohri,Sergeui Vorobiev,Alexander A Fedorov,Steven C Almo,Shoichiro Ono

doi:10.1074/jbc.m403351200

Abstract

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.

Highlights

Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin
We determined the crystal structure of Caenorhabditis elegans AIP1 (UNC-78), which revealed 14 WD40 modules arranged in two seven-bladed ␤-propeller domains
We determined the crystal structure of UNC78/AIP1 and identified five residues of UNC-78 that are involved in the disassembly of UNC-60B (ADF/cofilin)-bound actin filaments

Summary

PDB entry Space group Unit cell

Resolution of data (Å) Completeness of data set (%) Average I/␴ of data set Completeness of outermost shell (%) Average I/␴ of outermost shell Rsym(I)a (%) Phasing Phasing power at 2.5 Åb Overall figure of merit. In Caenorhabditis elegans, mutations of the unc-78 gene, which encodes AIP1, cause disorganization of actin filaments in the body wall muscle [21], and unc-78 shows genetic interactions with the unc-60B ADF/cofilin gene that is required for proper organization of muscle actin filaments [22]. Co-localization of AIP1 with ADF/cofilin is reported in other organisms (reviewed in Ref. 5), suggesting that they are evolutionarily conserved co-regulators for actin cytoskeleton. We determined the crystal structure of C. elegans UNC-78/ AIP1 at 1.9-Å resolution, which allowed for the identification of highly conserved surface residues. Mutagenesis studies targeted 20 of these residues and identified five residues that alter the activity of UNC-78/AIP1. These mutations uncouple filament disassembly activity from capping activity. Our results highlight the location of a functionally relevant surface of UNC-78/AIP1 and suggest distinct roles for filament binding and disassembly

EXPERIMENTAL PROCEDURES

TABLE II Mutagenesis primers

RESULTS

Wild type

DISCUSSION