The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins.

The complete amino acid sequence of muscle desmin reported here is the first for an intermediate filament protein. Alignment with partial data available for vimentin, glial fibrillary acid protein, neurofilament 68 K, two wool alpha-keratins, and a recently described DNA clone covering 90% of an epidermal keratin shows that all seven proteins have extensive homologies and therefore form a complex multigene family, the intermediate filament proteins. The hard alpha-keratins of wool appear to be a special subset of epithelial keratins. The sequence information reveals, as the dominant structural principle, a rod-like middle domain arising from several alpha-helical segments able to form interchain coiled-coil elements. The proposed helices are separated by short spacers, which like the two terminal domains seem built from non-alpha-helical material. Attention is drawn to the sometimes very striking sequence homologies along the rod and the high sequence variability in the terminal domains. Finally, chemical cross-linking experiments performed on the isolated desmin rod show that intermediate filament structure seems not to be based on triple-stranded coiled-coils as currently thought, but rather reflects protofilament units built as a dimer of normal interchain double-stranded coiled-coils.     

Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.     

Intermediate filaments: a historical perspective

Intracellular protein filaments intermediate in size between actin microfilaments and microtubules are composed of a surprising variety of tissue specific proteins commonly interconnected with other filamentous systems for mechanical stability and decorated by a variety of proteins that provide specialized functions. The sequence conservation of the coiled-coil, alpha-helical structure responsible for polymerization into individual 10 nm filaments defines the classification of intermediate filament proteins into a large gene family. Individual filaments further assemble into bundles and branched cytoskeletons visible in the light microscope. However, it is the diversity of the variable terminal domains that likely contributes most to different functions. The search for the functions of intermediate filament proteins has led to discoveries of roles in diseases of the skin, heart, muscle, liver, brain, adipose tissues and even premature aging. The diversity of uses of intermediate filaments as structural elements and scaffolds for organizing the distribution of decorating molecules contrasts with other cytoskeletal elements. This review is an attempt to provide some recollection of how such a diverse field emerged and changed over about 30 years.     

Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension

The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)     

The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation

Neurofilaments, assembled from NF-L, NF-M, and NF-H subunits, are the most abundant structural elements in myelinated axons. Although all three subunits contain a central, alpha-helical rod domain thought to mediate filament assembly, only NF-L self-assembles into 10-nm filaments in vitro. To explore the roles of the central rod, the NH2- terminal head and the COOH-terminal tail domain in filament assembly, full-length, headless, tailless, and rod only fragments of mouse NF-L were expressed in bacteria, purified, and their structure and assembly properties examined by conventional and scanning transmission electron microscopy (TEM and STEM). These experiments revealed that in vitro assembly of NF-L into bona fide 10-nm filaments requires both end domains: whereas the NH2-terminal head domain promotes lateral association of protofilaments into protofibrils and ultimately 10-nm filaments, the COOH-terminal tail domain controls lateral assembly of protofilaments so that it terminates at the 10-nm filament level. Hence, the two end domains of NF-L have antagonistic effects on the lateral association of protofilaments into higher-order structures, with the effect of the COOH-terminal tail domain being dominant over that of the NH2-terminal head domain. Consideration of the 21-nm axial beading commonly observed with 10-nm filaments, the approximate 21-nm axial periodicity measured on paracrystals, and recent cross-linking data combine to support a molecular model for intermediate filament architecture in which the 44-46-nm long dimer rods overlap by 1-3-nm head-to-tail, whereas laterally they align antiparallel both unstaggered and approximately half-staggered.     

Structural Basis for Stabilization of the Hypervariable D3 Domain of Salmonella Flagellin upon Filament Formation

The hypervariable D3 domain of Salmonella flagellin, composed of residues 190-283, is situated at the outer surface of flagellar filaments. A flagellin mutant deprived of the complete D3 domain (ΔD3_FliC) exhibited a significantly decreased thermal stability (T_m 41.9 °C) as compared to intact flagellin (T_m 47.3 °C). However, the stability of filaments formed from ΔD3_FliC subunits was virtually identical with that of native flagellar filaments. While D3 comprises the most stable part of monomeric flagellin playing an important role in the stabilization of the other two (D1 and D2) domains, the situation is reversed in the polymeric state. Upon filament formation, ordering of the disordered terminal regions of flagellin in the core part of the filament results in the stabilization of the radially arranged D1 and D2 domains, and there is a substantial increase of stability even in the distant outermost D3 domain, which is connected to D2 via a pair of short antiparallel β-strands. Our experiments revealed that crosslinking the ends of the isolated D3 domain through a disulfide bridge gives rise to a stabilization effect reminiscent of that observed upon polymerization. It appears that the short interdomain linker between domains D2 and D3 serves as a stabilization center that facilitates propagation of the conformational signal from the filament core to the outer part of filament. Because D3 is a largely independent part of flagellin, its replacement by heterologous proteins or domains might offer a promising approach for creation of various fusion proteins possessing polymerization ability.     

Structure of the Rad50 x Mre11 DNA repair complex from Saccharomyces cerevisiae by electron microscopy

The RAD50 gene of Saccharomyces cerevisiae is one of several genes required for recombinational repair of double-strand DNA breaks during vegetative growth and for initiation of meiotic recombination. Rad50 forms a complex with two other proteins, Mre11 and Xrs2, and this complex is involved in double-strand break formation and processing. Rad50 has limited sequence homology to the structural maintenance of chromosomes (SMC) family of proteins and shares the same domain structure as SMCs: N- and C-terminal globular domains separated by two long coiled-coils. However, a notable difference is the much smaller non-coil hinge region between the two coiled-coils. We report here a structural analysis of full-length S. cerevisiae Rad50, alone and in a complex with yeast Mre11 by electron microscopy. Our results confirm that yeast Rad50 does have the same antiparallel coiled-coil structure as SMC proteins, but with no detectable globular hinge domain. However, the molecule is still able to bend sharply in the middle to bring the two catalytic domains together, indicating that the small hinge domain is flexible. We also demonstrate that Mre11 binds as a dimer between the catalytic domains of Rad50, bringing the nuclease activities of Mre11 in close proximity to the ATPase and DNA binding activities of Rad50.     

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro

To investigate the sequences important for assembly of keratins into 10- nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.     

Intermediate filament structure.

In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.     

Molecular basis of the C-terminal tail-to-tail assembly of the sarcomeric filament protein myomesin

Sarcomeric filament proteins display extraordinary properties in terms of protein length and mechanical elasticity, requiring specific anchoring and assembly mechanisms. To establish the molecular basis of terminal filament assembly, we have selected the sarcomeric M-band protein myomesin as a prototypic filament model. The crystal structure of the myomesin C-terminus, comprising a tandem array of two immunoglobulin (Ig) domains My12 and My13, reveals a dimeric end-to-end filament of 14.3 nm length. Although the two domains share the same fold, an unexpected rearrangement of one beta-strand reveals how they are evolved into unrelated functions, terminal filament assembly (My13) and filament propagation (My12). The two domains are connected by a six-turn alpha-helix, of which two turns are void of any interactions with other protein parts. Thus, the overall structure of the assembled myomesin C-terminus resembles a three-body beads-on-the-string model with potentially elastic properties. We predict that the found My12-helix-My13 domain topology may provide a structural template for the filament architecture of the entire C-terminal Ig domain array My9-My13 of myomesin.     

Cytoskeletal cytoplasmic filament ribbon of Treponema: a member of an intermediate-like filament protein family

Development of genetic systems for many bacterial genera, including Treponema, now allow the study of structures that are specific to certain pathogens. The cytoplasmic filament ribbon of treponemes that is involved in the cell division cycle has a unique organization. Cytoplasmic bridging proteins connect the filaments, maintaining the distance between them and providing the overall ribbon-like structure. The filaments are anchored by proteins associated with the inner membrane. Each filament is composed of a unique monomer, the cytoplasmic filament protein A (CfpA), with coiled-coils secondary structures. CfpA is part of a growing family of proteins that we propose to call bacterial intermediate-like filaments (BILF).     

Myosin and paramyosin are organized about a newly identified core structure

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D. M. III, I. Ortiz, G. C. Berliner, and H. F. Epstein, 1983, Cell, 34:477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.     

Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin

We used chicken alpha spectrin as a ligand probe to isolate Drosophila beta spectrin cDNA sequences from a lambda gt11 expression library. Analysis of 800 residues of deduced amino acid sequence at the amino- terminal end revealed a strikingly conserved domain of integral of 230 residues that shows a high degree of sequence similarity to the amino- terminal domains of alpha actinin and dystrophin. This conserved domain constitutes a new diagnostic criterion for spectrin-related proteins and allows the known properties of one of these proteins to predict functional properties of the others. The conservation of the amino- terminal domain, and other regions in spectrin, alpha actinin, and dystrophin, demonstrates that a common set of domains were linked in different combinations through evolution to generate the distinctive members of the spectrin superfamily.     

Terminal assembly of sarcomeric filaments by intermolecular beta-sheet formation

The contraction-relaxation cycle of muscle cells translates into large movements of several filament systems in sarcomeres, requiring special molecular mechanisms to maintain their structural integrity. Recent structural and functional data from three filaments harboring extensive arrays of immunoglobulin-like domains - titin, filamin and myomesin--have, for the first time, unraveled a common function of their terminal domains: assembly and anchoring of the respective filaments. In each case, the protein-protein interactions are mediated by antiparallel dimerization modules via intermolecular beta-sheets. These observations on terminal filament assembly indicate an attractive model for several other filament proteins that require structural characterization.     

Evolution of keratin genes: Different protein domains evolve by different pathways

Summary Intermediate filaments are composed of a family of proteins that evolved from a common ancestor. The proteins consist of three domains: a central, alpha-helical domain similar in all intermediate filaments, bracketed by two domains that are variable in length and structure. Within the intermediate-filament family, several subfamilies have been recognized by immunologic and nucleic acid hybridization techniques. In this paper we present the sequence of the genomic DNA coding for a 65-kilodalton human keratin and compare it with the sequences of other intermediate-filament proteins. While the central, alpha-helical domains of these proteins show homologies that indicate a common ancestor, the sequences of the variable terminal domains indicate that the variable domains evolved through a series of tandem duplications and possibly by gene-conversion mechanisms.     

The Arg non-receptor tyrosine kinase modifies F-actin structure

The Arg (Abl-related gene) protein belongs to the Abl family of non-receptor tyrosine kinases that regulate cell motility and morphogenesis. It contains two actin-binding domains, one containing the talin-like I/LWEQ motif, and a C-terminal calponin homology (CH) domain. We used electron microscopy and single particle image analysis to reconstruct complexes of F-actin with full-length Arg, and fragments lacking either the I/LWEQ or CH domains. The Arg CH domain binds to actin's subdomain-1 (SD1) and induces a tilt of actin protomers. The I/LWEQ domain binds to either SD1 or SD4, closing the nucleotide binding cleft of actin. Although Arg can use either its CH or ILWEQ domains to bind an actin filament, both domains within Arg cannot bind simultaneously to adjacent protomers in the filament, consistent with its F-actin-bundling activity. The conformational changes in the filament introduced by Arg can explain the cooperative binding of Arg to F-actin and might prevent other actin binding proteins from binding to actin filaments.     

Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system.

Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.     

Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross- bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl- terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.