| Actin depolymerizing factor (ADF) and cofilin define a family of actin binding proteins essential for the rapid turnover of filamentous actin in vivo. Members of the ADF/cofilin family bind to both monomeric and filamentous actin and are regulated through a number of mechanisms, including phosphorylation, phosphoinositide binding, nuclear translocation, the state of the actin-bound nucleotide, and pH. Despite the availability of ADF/cofilin structures from several species, the structural bases for ADF/cofilin activity and regulation are unknown. To expand the species-dependent structural diversity available for the ADF/cofilin family, a structural investigation of ADF1 from Arabidopsis thaliana (AtADF1) was undertaken. Crystal structure determination of AtADF1 to 2.0 Å resolution reveals the unexpected presence of a bound lauryldimethylamine oxide (LDAO) molecule and the concomitant displacement and disordering of the C-terminal helix. To investigate the possible movement of the C-terminus with AtADF1:phosphoinositide complex formation, tryptophan fluorescence and circular dichroism experiments were carried out for AtADF1 in the presence and absence of various lipids. Changes in AtADF1 structure are apparent with the addition of phosphoinositides but not phosphatidylserine or inositol triphosphate. Although the precise elements undergoing a structural change upon phosphoinositide binding remain unclear, several candidate regions which would likely disrupt actin binding upon significant structural movement are suggested.; Structure determination of AtADF1 allows for a comparative structural analysis of the ADF/cofilin family. Superposition of the four ADF/cofilin isoform structures permits an accurate sequence alignment that differs from previously reported data with respect to the location of vertebrate-specific inserts, and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants and “other eukaryotes.” Within these groups, several structurally conserved residues have been identified which are not conserved in sequence across the ADF/cofilin family. Residues which are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in α-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding. |
