NMR solution structures of actin depolymerizing factor
Actin is one of the most conserved proteins in nature, differing by no more than 5% in species as diverse as algae and humans.1 The individual subunits of actin are known as globular actin (G-actin). G-actin spontaneously assembles to form a filamentous helical polymer, called F-actin. F-actin provides mechanical support to the cell, determines cell shape, forms the cytoskeleton, and enables cell movement, cytokinesis, morphogenesis, endocytosis, and cell division.1 Relative concentrations of F-actin and G-actin are regulated by a variety of intricate signaling pathways involving many proteins.
These include monomer sequestrant proteins, actin filament termination proteins, actin filament crossover proteins, filament nucleation and cutting proteins, and numerous small actin-binding proteins. Despite the diverse biochemical activities of these domains, there are relatively few actin-binding motifs. One of these motifs is presented by the homology domain of the actin depolymerization factor (ADF-H) of 15-20 kDa (∼150 amino acids) .
ADF-H domains are widely distributed: they are found in yeast, bacteria, and plants, and all eukaryotic cells appear to have at least one.5 ADF and cofilin are the founding members of what is now called the ADF-H family of domains . They were initially identified and named according to their ability to depolymerize F-actin (ADF) or form cofilamentous structures with actin (cofilin). They are very similar (eg actin dynamics) 6 and lack clearly distinctive biochemical properties.
Therefore, they are often thought of as a single entity: ADF / cofilin.1 However, they are the product of different genes: most vertebrates have one gene encoding ADF and two genes encoding cofilin (muscle cofilins and not muscular). Many lower eukaryotes, such as D. melanogaster and S. cerevisiae, have only one gene. Genes often have introns and generate several different protein products; those without introns are likely pseudogenes.5 Eukaryotic ADFs / cofilins share approximately 40% sequence identity.2 Mutations that inactivate cofilin / ADF are lethal in C. elegans, D. melanogaster, and S. cerevisiae.
With the advent of cDNA cloning and sequencing, there have been many additions to the ADF-H family of domains based on sequence homology. Phylogenetically (Fig. 3), 7 family members can be divided into five functionally distinct classes: I-cofilin / ADF, II-glial maturation factor (GMF), III-coactosin, IV-twinfilin and V-Abp1 / drebrin. They have diverse sequences (Figures 1 and 2) and share approximately 20% sequence identity at the amino acid level1,2. Very little is known about ADF-H domains other than ADF / cofilin. They differ in their ability to bind G and F actin (Table I). All five classes can be found in species as diverse as H. sapiens, M. musculus, D. melanogaster, and D. discoideum.
Two types of GMF (Class II) have been identified in mice and humans: GMF β was identified as a nerve growth factor, involved in the development of the nervous system, 8 while GMF γ was initially identified as a molecule with high similarity to GMF β, 9 although it is not expressed in brain, neuronal or glial cells, but in microvascular endothelial and inflammatory cells10. 11
Drebrins (Class V) contain an N-terminal ADF-H domain and a C-terminal SH3 domain that binds to dynamin. 12 Human HIP-55-drebrin-like protein is important for immune system function and is regulated by T-cell antigen receptor (TCR) signaling .13 Few ADF-H domain structures of classes other than Class I (ADF / cofilin) have been reported (Table I) .14-18 We now report the NMR structures in solution of five domains of these four underrepresented classes is: mouse GMF β and γ (Class II), similar to mouse coactosin (Class III), the C-terminal domain (residues 161-313) of the mouse twinfilin 1 (Class IV) and the ADF-H domain of human HIP -55 drebrin type (Class V).
The latter domain is the first published structure of a mammalian drebrin-like domain (the structure of the S. cerevisiae Abp1 ADF-H domain was previously reported) .18 The goal is to deduce differences in F and G actin binding. of the structures and identify the common characteristics of each ADF-H class.