To date, three high-resolution structures of ADF homology domains have been reported. The structure of human destrin was determined by nuclear magnetic resonance (Hatanaka et al., 1996
) and was followed by the 2.3-Å resolution x-ray crystal structures of yeast cofilin (Fedorov et al., 1997
) and Acanthamoeba
actophorin (Leonard et al., 1997
). All three structures display a very similar fold, with a central six-stranded mixed β-sheet sandwiched between two pairs of α-helices, one on each face (Figure a). The only significant difference between these structures is the presence of an additional α-helix in human destrin between β-strands 1 and 2 (Figures and a).
Figure 4 (a) Ribbon diagram of the yeast cofilin structure. Cofilin has a central mixed β-sheet, which is sandwiched between two pairs of α-helices. The positions of the insertions in mammalian cofilins are in green and blue, and the diverged region (more ...)
A surprising and remarkable feature of the three-dimensional structures of ADF/cofilin proteins is the similarity of their overall fold to another actin-binding module, the gelsolin homology domain. Members of both actin-binding protein families are built around a central mixed β-sheet and show the same topological connections of secondary structure elements (McLaughlin et al., 1993
; Hatanaka et al., 1996
Because gelsolin consists of multiple segments, and because it appears that different gelsolin segments interact with actin through different interfaces, it is possible that at least one of the gelsolin segments interacts with actin in a manner similar to that of ADF/cofilin proteins. In support of this hypothesis, recent studies have suggested that cofilin and gelsolin segment 2 might interact with actin in a similar manner (Van Troys et al., 1997
). In contrast, it appears that the actin-binding surfaces of cofilin and gelsolin segment 1 are significantly different from each other. The gelsolin segment 1–actin interface has been identified from x-ray crystallography studies (McLaughlin et al., 1993
). Systematic mutagenesis studies of yeast cofilin suggested that the actin-binding site of cofilin is more extended than the actin-binding site of gelsolin segment 1 and is not as clearly built around the “long” α-helix (α-3 in yeast cofilin) (Lappalainen et al., 1997
; Figure B). Furthermore, electron microscopy studies on cofilin-decorated actin filaments indicate that the cofilin-binding site on an actin filament is significantly different from the gelsolin segment 1-binding site (McGough et al., 1997
). Finally, it should be noted that whereas gelsolin segment 1 is able to interact only with actin monomers (Weeds and Maciver, 1993
), members of the ADF/cofilin class of proteins interact tightly with both actin monomers and actin filaments.
Based on the structural homology and the similarity of their biochemical activities (actin binding), it has been proposed that ADF/cofilin proteins and gelsolins have evolved from a common ancestral actin-binding protein (Hatanaka et al., 1996
). Primary sequence alignments of ADF/cofilins with gelsolins, however, do not reveal sequence identity between these groups of proteins higher than would be expected between two unrelated proteins. The inclusion of gelsolins in the ADF-H protein family is therefore not justified at this stage, and a meaningful phylogenetic analysis that included gelsolin would be impossible. It must be noted that convergent, as well as divergent, evolution could have given rise to the structural similarities between the proteins. For these reasons, we have in this essay focused only on the three classes of ADF-H domain proteins that clearly evolved from a common ancestral gene.
From the sequence alignment shown in Figure and the structure in Figure a, it can be predicted that all ADF-H domains have a similar overall fold. Although, for example, the sequence homology between members of the drebrin/Abp1 and ADF/cofilin classes is low (13–15% identity within and between species), the predicted secondary structure elements identified in the ADF-H domain structural models are well conserved throughout the entire family. Furthermore, predicted loop regions connecting secondary structure elements are less conserved, and insertions are located in the predicted loop regions. Finally, the hydrophobic core elements are well conserved (see below; Figure a, residues indicated in yellow).
The sequence alignment also suggests that all ADF-H domains probably interact with actin through a similar interface. The residues essential for the interaction of yeast cofilin with actin (Figures , asterisks, and 4b, red) are relatively well conserved in all three classes of ADF-H domain proteins. More specifically, residues Arg96
, and Glu126
(numbering based on positions in yeast cofilin), which are essential for actin monomer and actin filament binding in yeast cofilin (Lappalainen et al., 1997
), show extremely high conservation throughout the three protein families. Furthermore, the five N-terminal residues of yeast cofilin, which have been shown to be essential for actin monomer and filament binding, are relatively well conserved in the ADF-H domains (Lappalainen et al., 1997
). The most highly conserved residues in this region are Ser4
(numbering based on positions in yeast cofilin), suggesting that these two residues might form an important actin-binding sequence within the N-terminal region of ADF-H domains (the first five residues of cofilin are not shown in Figure , because they were found to be disordered in the x-ray crystal structure determination). Interestingly, in vertebrate ADF/cofilin proteins, interactions with actin can be down-regulated by phosphorylation of the aforementioned serine (Agnew et al., 1995
). The suggestion that the F-actin interface is conserved, at least between the ADF/cofilin and drebrin/Abp1 classes, is also supported by biochemical data that show that both ADF (Bernstein and Bamburg, 1982
) and drebrin (Ishikawa et al., 1994
) compete with tropomyosin for actin filament binding.
In addition to the highly conserved residues that have already been shown to be important for the interaction between cofilin and actin, there are also a number of other residues that are well conserved throughout ADF homology domains. These residues fall approximately into two different categories. The first category consists of residues located in the hydrophobic core of cofilin, and these therefore appear to be important for protein stability or correct folding. These residues include Tyr64
, and Tyr101
(numbers based on positions in yeast cofilin; Figure a, yellow). A critical role of maize ADF residues Tyr82
(corresponding to residues Tyr64
in yeast cofilin) in proper protein folding has recently been demonstrated by site-directed mutagenesis (Jiang et al., 1997
). The second category of highly conserved residues are those that, because they are exposed on the surface of the protein, may be involved in protein–protein interactions. All of these residues (Met99
, and Gly114
) except Ser45
are located close to the actin-binding site as identified by Lappalainen et al., (1997)
, indicating that these residues might take part in interactions with actin (Figure b). Ser45
is located at the opposite side of the molecule. Because this residue is at the end of the β-strand-3 and because this position is in most cases occupied by either serine or glycine, it is possible that a residue lacking a bulky side chain is required at this position for steric reasons. Gln120
, which is conserved in all but one of the ADF/cofilin proteins and is replaced by asparagine in drebrins and mSH3P7, but which is not conserved in twinfilins, coactosin, and depactin, is also located close to the actin-binding site of cofilin.
An interesting and instructive sequence (and, by inference, structural) variance among the ADF-H domains is revealed by the alignment. There are two insertions present in the vertebrate ADF/cofilins but not in any other members of the ADF-H domain family. The first insertion is located between α-helix 1 and β-strand-2, and the second insertion is between β-strand-2 and β-strand-3 (Figures and a, green and blue loops, respectively). The first insertion forms a short α-helix in human destrin and is always followed by a putative nuclear localization signal (KKRKK) found only in vertebrate ADF/cofilin proteins (Figure ; note that human destrin-2 is a pseudogene and contains the sequence KKRTK in this region). Nuclear localization of ADF/cofilin has been demonstrated in mammalian cells placed under stress (Ohta et al., 1989
). Conceivably, these insertions may play a role in a nuclear function of mammalian cofilins. It is important, however, to note that an identical KKRKK sequence is also found in ActA, a protein that plays central role in the actin-based motility of the intracellular pathogen Listeria monocytogenes
. Mutagenesis studies have shown that this lysine-rich sequence in ActA plays an important role in actin filament nucleation (Lasa et al., 1997
). It is therefore formally possible that this sequence might have a similar role in mammalian cofilins.
A second structural difference within the ADF-H domain family revealed by our sequence alignment is that β-strand-4 and β-strand-5 within the drebrin/Abp1 class are shortened and/or disrupted by prolines. The cluster of three charged residues preceding Lys81 (based on position in S. cerevisiae) found in all mammalian and avian cofilins is replaced by hydrophobic but flexible amino acids in the drebrin/Abp1 class. Thus, it seems that the rigid handle protruding from the main body of ADF/cofilin is shortened in the entire drebrin/Abp1 family and probably has a different tertiary structure, the functional implications of which need to be explored.
Finally, our sequence alignment shows that the C-terminal regions following β-strand-6 in all twinfilin ADF-H domains are longer and align poorly with other members of ADF-H domain family (Figure ). It is possible that this region imparts a unique function to these proteins, such as the reported kinase activity (Beeler et al., 1994
). Alternatively, the extension that separates the two tandem ADF-H domains in twinfilin might serve as a linker, thereby conferring an appropriate spatial relationship between the two domains.