A comparative and phylogenetic analysis of the alpha-actinin rod domain

Alpha-actinin is a ubiquitous actin-binding protein, composed of 3 domains; an actin-binding domain and a calcium-binding domain at the termini, connected by a rod domain composed by 1, 2, or 4 spectrin repeats (SRs). To understand how the rod domain has evolved during evolution, we have analyzed and compared the amino acid residue heterogeneity and phylogeny of the SRs of alpha-actinins of vertebrates, invertebrates, fungi, and several protozoa. The repeats of vertebrate alpha-actinins show a high degree of similarity, whereas repeats of invertebrates, fungi, and, in particular, of protozoa are more divergent. In the phylogeny, SR1 of all species were clustered together, independent of the number of repeats in the protein. It was also obvious that the second and last repeat in fungi (SR2) grouped with the fourth and last repeat of vertebrates and invertebrates (SR4). Therefore, the phylogeny implied that the rod domain of the cenancestral alpha-actinin only contained one SR. It was also obvious that SR2 of fungi are related to SR4 of vertebrates and invertebrates, implying that in the second intragenic duplication 2 repeats (i.e., what become SR2 and SR3) were inserted between the initial 2 repeats that become SR1 and SR4.     

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.     

Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.     

The α-Actinin Gene Family: A Revised Classification

The sequencing of a genome is the first stage of its complete characterization. Subsequent work seeks to utilize available sequence data to gain a better understanding of the genes which are found within a genome. Gene families comprise large portions of the genomes of higher vertebrates, and the available genomic data allow for a reappraisal of gene family evolution. This reappraisal will clarify relatedness within and between gene families. One such family, the alpha-actinin gene family, is part of the spectrin superfamily. There are four known loci, which encode alpha-actinins 1, 2, 3, and 4. Of the eight domains in alpha-actinin, the actin-binding domain is the most highly conserved. Here we present evidence gained through phylogenetic analyses of the highly conserved actin-binding domain that alpha-actinin 2 was the first of the four alpha-actinins to arise by gene duplication, followed by the divergence of alpha-actinin 3 and then alpha-actinins 1 and 4. Resolution of the gene tree for this gene family has allowed us to reclassify several alpha-actinins which were previously given names inconsistent with the most widely accepted nomenclature for this gene family. This reclassification clarifies previous discrepancies in the public databases as well as in the literature, thus eliminating confusion caused by continued misclassification of members of the alpha-actinin gene family. In addition, the topology found for this gene family undermines the 2R hypothesis theory of two rounds of genome duplication early in vertebrate evolution.     

Unfolding a linker between helical repeats

In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including alpha-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit water. The results for alpha-actinin repeats reveal rate-dependent pathways, with one pathway showing that the linker between repeats unfolds, which may explain the single-repeat unfolding pathway observed in AFM experiments. A second pathway preserves the structural integrity of the linker, which explains the tandem-repeat unfolding event. Unfolding of the linker begins with a splay distortion of proximal loops away from hydrophobic contacts with the linker. This is followed by linker destabilization and unwinding with increased hydration of the backbone. The end result is an unfolded helix that mechanically decouples tandem repeats. Molecularly detailed insights obtained here aid in understanding the mechanical coupling of domain stability in spectrin family proteins.     

Independent movement, dimerization and stability of tandem repeats of chicken brain alpha-spectrin

Previous X-ray crystal structures have shown that linkers of five amino acid residues connecting pairs of chicken brain alpha-spectrin and human erythroid beta-spectrin repeats can undergo bending without losing their alpha-helical structure. To test whether bending at one linker can influence bending at an adjacent linker, the structures of two and three repeat fragments of chicken brain alpha-spectrin have been determined by X-ray crystallography. The structure of the three-repeat fragment clearly shows that bending at one linker can occur independently of bending at an adjacent linker. This observation increases the possible trajectories of modeled chains of spectrin repeats. Furthermore, the three-repeat molecule crystallized as an antiparallel dimer with a significantly smaller buried interfacial area than that of alpha-actinin, a spectrin-related molecule, but large enough and of a type indicating biological specificity. Comparison of the structures of the spectrin and alpha-actinin dimers supports weak association of the former, which could not be detected by analytical ultracentrifugation, versus strong association of the latter, which has been observed by others. To correlate features of the structure with solution properties and to test a previous model of stable spectrin and dystrophin repeats, the number of inter-helical interactions in each repeat of several spectrin structures were counted and compared to their thermal stabilities. Inter-helical interactions, but not all interactions, increased in parallel with measured thermal stabilities of each repeat and in agreement with the thermal stabilities of two and three repeats and also partial repeats of spectrin.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

Primary structure of chicken skeletal muscle and fibroblast alpha-actinins deduced from cDNA sequences

The complete 897-amino-acid sequence of chicken skeletal muscle alpha-actinin and the 856-amino-acid sequence (97% of the entire sequence) of chicken fibroblast alpha-actinin have been determined by cloning and sequencing the cDNAs. Genomic Southern analysis with the cDNA sequences shows that skeletal and fibroblast alpha-actinins are encoded by separate single-copy genes. RNA blot analyzes show that the skeletal alpha-actinin gene is expressed in the pectoralis muscle and that the fibroblast gene is expressed in the gizzard smooth muscle as well as in the fibroblast. The deduced skeletal alpha-actinin molecule has a calculated Mr of 104 x 10(3), and each alpha-actinin can be divided into three domains: (1) the NH2-terminal highly conserved actin-binding domain, which shows similarity to the product of the Duchenne's muscular dystrophy locus; (2) the middle rod-shaped dimer-forming domain, which contains the spectrin-type repeat units; and (3) the COOH-terminal two EF-hand consensus regions. Comparison of the skeletal alpha-actinin sequence with the fibroblast and smooth muscle alpha-actinin sequences demonstrated that the EF-hand structure was conserved in all of these alpha-actinin sequences, despite the reported variability of the Ca2+ sensitivities of the actin-gelation by various alpha-actinin isoforms.     

Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site.

The antiparallel side-to-side association of spectrin alpha and beta monomers is a two-step process which occurs in seconds even at 0 degrees C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homologous segments or repeats. Three repeats in the nucleation site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in alpha-actinin. The adjacent actin binding domain on the beta subunit and the adjacent EF hand motifs on the alpha subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both subunits. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Heterozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.     

Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein

The complete sequence of the human Duchenne muscular dystrophy (DMD) cDNA has been determined. The 3685 encoded amino acids of the protein product, dystrophin, can be separated into four domains. The 240 amino acid N-terminal domain has been shown to be conserved with the actin-binding domain of alpha-actinin. A large second domain is predicted to be rod-shaped and formed by the succession of 25 triple-helical segments similar to the repeat domains of spectrin. The repeat segment is followed by a cysteine-rich segment that is similar in part to the entire COOH domain of the Dictyostelium alpha-actinin, while the 420 amino acid C-terminal domain of dystrophin does not show any similarity to previously reported proteins. The functional significance of some of the domains is addressed relative to the phenotypic characteristics of some Becker muscular dystrophy patients. Dystrophin shares many features with the cytoskeletal protein spectrin and alpha-actinin and is a large structural protein that is likely to adopt a rod shape about 150 nm in length.     

Alpha-actinin expression during avian myogenesis in vivo. Evidence for the existence of an embryo-specific isoform of alpha-actinin

The isoforms of skeletal muscle alpha-actinin present during chick embryogenesis were analyzed by two-dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8-15 days contain an embryo-specific isoform of alpha-actinin. The embryonic alpha-actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of alpha-actinin were 100 kDa and 5.85, respectively. To characterize the two classes of alpha-actinin polypeptides we have compared the two proteins by 125I-labeled two-dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of alpha-actinin. The developmental sequence of the expression of the alpha-actinins was also studied. In extracts of skeletal muscle from 8-10-day-old embryos, only the embryonic isoform was detected. In extracts from 15-day-old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected. These data suggested that the embryonic and the adult isoform of alpha-actinins are distinct proteins and that during skeletal myogenesis in ovo one class of alpha-actinin is replaced by a new class of alpha-actinin polypeptides, and that the latter is maintained into adulthood.     

Polymorphism of alpha-actinin. Electrophoretic and immunological studies of rabbit skeletal muscle alpha-actinins

Heterogeneity of alpha-actinins from rabbit skeletal muscles was studied. Polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate has made it possible to distinguish two closely related alpha-actinins from rabbit fast, white muscle. One isoprotein (designated type I alpha-actinin) appears to be preferentially located in the psoas muscle, while the other (designated type II alpha-actinin) appears to be preferentially located in the longissimus dorsi muscle. Electrophoretic analyses have further shown that the two isoproteins are present as mixtures in most rabbit white, fast-twitch muscles. A standard polyacrylamide gel--sodium dodecyl sulfate/polyacrylamide gel sequential electrophoretic procedure was developed to resolve the different alpha-actinin dimers and to determine their subunit compositions. By this technique, both type I and type II alpha-actinins appeared to be homodimers. No heterodimeric species of alpha-actinin were detected. alpha-Actinin of red, slow-twitch muscles was similar to type II alpha-actinin of fast, white muscle on one-dimensional and two dimensional gels. However, slow, red muscle alpha-actinin was significantly different from fast, white muscle alpha-actinins in terms of one-dimensional peptide mapping and immunological cross-reactivity.     

Maintaining epithelial integrity: a function for gigantic spectraplakin isoforms in adherens junctions

The Short stop (Shot/Kakapo) spectraplakin is a giant cytoskeletal protein, which exists in multiple isoforms with characteristics of both spectrin and plakin superfamilies. Previously characterized Shot isoforms are similar to spectrin and dystrophin, with an actin-binding domain followed by spectrin repeats. We describe a new large exon within the shot locus, which encodes a series of plakin repeats similar to the COOH terminus of plakins such as plectin and BPAG1e. We find that the plakin repeats are inserted between the actin-binding domain and spectrin repeats, generating isoforms as large as 8,846 residues, which could span 400 nm. These novel isoforms localized to adherens junctions of embryonic and follicular epithelia. Loss of Shot within the follicle epithelium leads to double layering and accumulation of actin and ZO-1 in between, and a reduction of Armadillo and Discs lost within, mutant cells, indicative of a disruption of adherens junction integrity. Thus, we identify a new role for spectraplakins in mediating cell-cell adhesion.     

MACF1 gene structure: a hybrid of plectin and dystrophin

Mammalian MACF1 (Macrophin1; previously named ACF7) is a giant cytoskeletal linker protein with three known isoforms that arise by alternative splicing. We isolated a 19.1-kb cDNA encoding a fourth isoform (MACF1-4) with a unique N-terminus. Instead of an N-terminal actin-binding domain found in the other three isoforms, MACF1-4 has eight plectin repeats. The MACF1 gene is located on human Chr 1p32, contains at least 102 exons, spans over 270 kb, and gives rise to four major isoforms with different N-termini. The genomic organization of the actin-binding domain is highly conserved in mammalian genes for both plectin and BPAG1. All eight plectin repeats are encoded by one large exon; this feature is similar to the genomic structure of plectin. The intron positions within spectrin repeats in MACF1 are very similar to those in the dystrophin gene. This demonstrates that MACF1 has characteristic features of genes for two classes of cytoskeletal proteins, i.e., plectin and dystrophin. Received: 24 April 2001 / Accepted: 29 June 2001     

Brush-border alpha-actinin? Comparison of two proteins of the microvillus core with alpha-actinin by two-dimensional peptide mapping

The bundle of filaments within the intestinal microvillus contains four major polypeptides in addition to actin calmodulin, a 70-kdalton subunit and two polypeptides with molecular masses similar to that of the Z-line component alpha-actinin (95 and 105 kdaltons). Two- dimensional mapping of tryptic peptides indicates that (a) alpha- actinins from chicken skeletal, cardiac, and smooth muscle are similar but not identical proteins and that skeletal alpha-actinin in more similar to the cardiac subunit than to the alpha-actinin from gizzard; (b) the brush-border 95- and 105-kdalton subunits are closely related to each other, but the smaller subunit is not a proteolytic fragment of the 105-kdalton subunit; and (c) although there is considerable peptide overlap between the brush-border subunits and the three alpha-actinins, the peptide maps of the 95- and 105-kdalton proteins are substantially distinct from the various alpha-actinin maps, suggesting that neither brush-border subunit is a bona fide alpha-actinin. Nevertheless, on the basis of peptide mapping criteria alone, one cannot exclude the possibility that the brush-border subunits are "alpha-actinin-like." However, there is no immunological cross-reactivity between the brush- border subunits and alpha-actinins, using antibodies prepared against gizzard alpha actinin.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

The structure of a tandem pair of spectrin repeats of plectin reveals a modular organization of the plakin domain

Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin alpha6beta4 in hemidesmosomes. The structure is formed by two spectrin repeats connected by an alpha-helix that spans these two repeats. While the first repeat is very similar to other known structures, the second repeat is structurally different with a hydrophobic core, narrower than that in canonical spectrin repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive spectrin repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central spectrin repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.