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 One mutant, zmadf3-1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3.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 ␣-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 Å, respectively, with corresponding bond angles of 99.5°and 113°, close to the optimum for a strong hydrogen bond.

Plant Actin-Depolymerizing Factors Possess Opposing Biochemical Properties Arising from Key Amino Acid Changes throughout Evolution

“…The basic residues found in b-sheet 4, b-sheet 5, a-helix 3, and a-helix 4 are conserved in several ADF/cofilin genes and are important for F-actin binding activity (Jiang et al, 1997;Lappalainen and Drubin, 1997;Pope et al, 2000;. According to published reports, b-sheets 4 and 5 and a-helix 4 of ADF are in spatial proximity to one another and likely form an essential F-actin binding surface (F-surface 1) with a relatively high level of conservation of key basic residues (Lys-86, Lys-88, and Arg-141) ( Figure 5A) (Lappalainen and Drubin, 1997;Bowman et al, 2000;Pope et al, 2000).…”

The C-terminal Tail of UNC-60B (Actin Depolymerizing Factor/Cofilin) Is Critical for Maintaining Its Stable Association with F-actin and Is Implicated in the Second Actin-binding Site

“…Two regions have been implicated in filament binding as follows: (i) a cluster of charged residues (15,27) in a loop connecting ␤2 and ␤3 in destrin and at a similar position at the beginning of ␤5 in yeast cofilin, and (ii) a group of charged residues in helix ␣4 in yeast cofilin (27). In addition, mutations of conserved tyrosine residues (Tyr 67 and Tyr 70 in maize ADF3) in the apolar core of the molecule results in uncoupling of monomer and filament binding activities (37).…”

The Identification of a Second Cofilin Binding Site on Actin Suggests a Novel, Intercalated Arrangement of F-actin Binding

“…The cofilin-actin interface has been investigated by a number of methods including chemical cross-linking (19,(55)(56)(57)(58), competition with other ABPs (24, 59 -63), structural prediction analysis (63), and mutagenesis (64,65). The consensus of these studies is that subdomain 1 of actin is the principal actinbinding interface that binds the long helix of the ADF-cofilin.…”

“…These authors suggested that one of these sites bound one actin monomer within a filament, whereas the second site bound a second actin monomer. Another mutagenic study (64) suggests the importance of the "long helix" (␣3) stability in binding F-actin, but not G-actin.…”