title: F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF).
abstract: Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3-1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3-2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long alpha-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 A, respectively, with corresponding bond angles of 99.5 degrees and 113 degrees, close to the optimum for a strong hydrogen bond.
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Jiang
authors.forename: A G
authors.initials: AG
authors.lastname: Weeds
authors.forename: S
authors.initials: S
authors.lastname: Khan
authors.forename: P J
authors.initials: PJ
authors.lastname: Hussey
mesh_terms: D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence; D003598:Cytoskeletal Proteins; D051345:Destrin; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D014443:Tyrosine / Q000235:genetics; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C495824:DSTN protein, human; D051345:Destrin; D010940:Plant Proteins; D014443:Tyrosine