Structure of the Intermediate Filament-Binding Region of Desmoplakin

Desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of the full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal ~100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with R_g = 51.5 Å and D_max = 178 Å. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions.     

Molecular mechanism of intermediate filament recognition by plakin proteins

The plakin family of cytolinkers interacts with intermediate filaments (IFs) through plakin repeat domain (PRD) and linker modules. Recent structure/function studies have established the molecular basis of envoplakin-PRD and periplakin-linker interactions with vimentin. Both plakin modules share a broad basic groove which recognizes acidic rod elements on IFs, a mechanism that is applicable to other plakin family members. This review postulates a universal IF engagement mechanism that illuminates the specific effects of pathogenic mutations associated with diseases including arrhythmogenic right ventricular cardiomyopathy, and reveals how diverse plakin proteins offer tailored IF tethering to ensure stable, dynamic and regulated cellular structures.     

Cutting edge: integration of human T lymphocyte cytoskeleton by the cytolinker plectin.

Chemokine-induced polarization of lymphocytes involves the rapid collapse of vimentin intermediate filaments (IFs) into an aggregate within the uropod. Little is known about the interactions of lymphocyte vimentin with other cytoskeletal elements. We demonstrate that human peripheral blood T lymphocytes express plectin, an IF-binding, cytoskeletal cross-linking protein. Plectin associates with a complex of structural proteins including vimentin, actin, fodrin, moesin, and lamin B in resting peripheral blood T lymphocytes. During chemokine-induced polarization, plectin redistributes to the uropod associated with vimentin and fodrin; their spatial distribution indicates that this vimentin-plectin-fodrin complex provides a continuous linkage from the nucleus (lamin B) to the cortical cytoskeleton. Overexpression of the plectin IF-binding domain in the T cell line Jurkat induces the perinuclear aggregation of vimentin IFs. Plectin is therefore likely to serve as an important organizer of the lymphocyte cytoskeleton and may regulate changes of lymphocyte cytoarchitecture during polarization and extravasation.     

An epidermal plakin that integrates actin and microtubule networks at cellular junctions

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.     

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.     

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A-2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.     

Deletion mutagenesis of the amino-terminal head domain of vimentin reveals dispensability of large internal regions for intermediate filament assembly and stability

Previous studies have shown that the non-alpha-helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino-terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25-38, 44-95, or 40-95 polymerized into wild-type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44-69 or 25-63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25-68, 44-103, or 88-103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non-alpha-helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non-alpha-helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non-alpha-helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C-terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as-yet-unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild-type vimentin.     

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.     

Self-assembly incompetence of synemin is related to the property of its head and rod domains

The mechanisms regulating the intermediate filament (IF) protein assembly are complex and not yet fully understood. All vertebrate cytoplasmic IF proteins have a central alpha-helical rod domain flanked by variable head and tail domains. The IF protein synemin cannot homopolymerize to form filament networks; it needs an appropriate copolymerization partner. To elucidate the roles of the vimentin head domain, the TAAL motif in the 2A region, and the TYRKLLEGEE motif in the 2B region of the rod domain in synemin filament formation, we have prepared a series of synemin constructs by site-directed mutagenesis and chimeric synemins having the vimentin head domain. The assembly properties of synemin constructs were assessed by the immunofluorescence of transient transfection into cultured SW13 cells without endogenous IFs. Our data showed that the formation of a filamentous network required at least the vimentin-like head domain and both the 2A and 2B regions of the rod domain.     

The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family with cytoskeletal linker properties. Mutations in BPAG1 cause sensory neuron degeneration and skin fragility in mice. We have analyzed the BPAG1 locus in detail and found that it encodes different interaction domains that are combined in tissue-specific manners. These domains include an actin-binding domain (ABD), a plakin domain, a coiled coil (CC) rod domain, two different potential intermediate filament-binding domains (IFBDs), a spectrin repeat (SR)-containing rod domain, and a microtubule-binding domain (MTBD). There are at least three major forms of BPAG1: BPAG1-e (302 kD), BPAG1-a (615 kD), and BPAG1-b (834 kD). BPAG1-e has been described previously and consists of the plakin domain, the CC rod domain, and the first IFBD. It is the primary epidermal BPAG1 isoform, and its absence that is the likely cause of skin fragility in mutant mice. BPAG1-a is the major isoform in the nervous system and a homologue of the microtubule actin cross-linking factor, MACF. BPAG1-a is composed of the ABD, the plakin domain, the SR-containing rod domain, and the MTBD. The absence of BPAG1-a is the likely cause of sensory neurodegeneration in mutant mice. BPAG1-b is highly expressed in muscles, and has extra exons encoding a second IFBD between the plakin and SR-containing rod domains of BPAG1-a.     

Epiplakin, a novel member of the Plakin family originally identified as a 450-kDa human epidermal autoantigen. Structure and tissue localization

A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serum from an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shown that it contains 5065 amino acids and has a molecular mass of 552 kDa. As reported previously this protein, which we call epiplakin, belongs to the plakin family, but it has some very unusual features. Epiplakin has 13 domains that are homologous to the B domain in the COOH-terminal region of desmoplakin. The last five of these B domains, together with their associated linker regions, are particularly strongly conserved. However, epiplakin lacks a coiled-coil rod domain and an amino-terminal domain, both of which are found in all other known members of the plakin family. Furthermore, no dimerization motif was found in the sequence. Thus, it is likely that epiplakin exists in vivo as a single-chain structure. Epitope mapping experiments showed that the original patient's serum recognized a sequence unique to epiplakin, which was not found in plectin. Immunofluorescence staining revealed the presence of epiplakin in whole sheets of epidermis and esophagus, in glandular cells of eccrine sweat and parotid glands and in mucous epithelial cells in the stomach and colon.     

Association of Mitochondria with Plectin and Desmin Intermediate Filaments in Striated Muscle

Plectin (M(r) > 500,000) is a versatile and widely expressed cytolinker protein. In striated muscle it is predominantly found at the Z-disc level where it colocalizes with the intermediate filament protein desmin. Both proteins show altered labeling patterns in tissues of muscular dystrophy patients. Moreover, mutations in the plectin gene lead to the autosomal recessive human disorder epidermolysis bullosa simplex with muscular dystrophy, and defects in the desmin gene have been shown to cause familiar cardiac and skeletal myopathy. Since intermediate filaments (IFs) in striated muscle tissue have been found to be intimately associated with mitochondria, we investigated whether plectin is involved in this association. Using postembedding immunogold labeling of Lowicryl sections and immunogold labeling of ultrathin cryosections, we show that plectin is associated with desmin IFs linking myofibrils to mitochondria at the level of the Z-disc and along the entire length of the sarcomere. The localization of plectin label at the mitochondrial membrane itself was consistent with a putative linker function of plectin between desmin IFs and the mitochondrial surface. In mitochondrion-rich muscle fibers, both plectin and desmin were part of an ordered arrangement of mitochondrial side branches, which wound around myofibrils adjacent to the Z-discs and were anchored into a filamentous network transversing from one fibril to the other. The association of mitochondria with plectin and IFs was seen also in tissues without regular distribution patterns of mitochondria, such as heart muscle and neonatal skeletal muscle tissues. These data were supplemented with in vitro binding assays showing direct interaction of plectin with desmin via its carboxy-terminal IF-binding domain. As a cytolinker protein associated with mitochondria and desmin IFs, plectin could play an important role in the positioning and shape formation, in particular branching, of mitochondrial organelles in striated muscle tissues.     

Crystal structure of a rigid four-spectrin-repeat fragment of the human desmoplakin plakin domain

The plakin protein family serves to connect cell-cell and cell-matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175-630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.     

The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.     

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.     

The nonlinear structure of the desmoplakin plakin domain and the effects of cardiomyopathy-linked mutations

Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. The desmoplakin N-terminal region is a 1056-amino-acid sequence of unknown structure. It mediates interactions with other desmosomal proteins, is found in a variety of plakin proteins, and spans what has been termed the “plakin domain,” which includes residues 180-1022 and consists of six spectrin repeats (SRs) and an Src homology 3 domain. Herein we elucidate the architecture of desmoplakin's plakin domain, as well as its constituent tandem SRs. Small-angle X-ray scattering analysis shows that the entire plakin domain has an “L” shape, with a long arm and a short arm held at a perpendicular angle. The long arm is 24.0 nm long and accommodates four stably folded SRs arranged in tandem. In contrast, the short arm is 17.9 nm in length and accommodates two independently folded repeats and an extended C-terminus. We show that mutations linked to arrhythmogenic right ventricular cardiomyopathy (K470E and R808C) cause local conformational alterations, while the overall folded structure is maintained. This provides the first structural and mechanistic insights into an entire plakin domain and provides a basis for understanding the critical role of desmoplakin in desmosome function.     

Recent insight into intermediate filament structure

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal ‘head’ and C-terminal ‘tail’ domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans.     

Interaction of the bullous pemphigoid antigen 1 (BP230) and desmoplakin with intermediate filaments is mediated by distinct sequences within their COOH terminus

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.     

Modeling the self-organization property of keratin intermediate filaments

Keratin intermediate filaments (IFs) fulfill an important function of structural support in epithelial cells. The necessary mechanical attributes require that IFs be organized into a crosslinked network and accordingly, keratin IFs are typically organized into large bundles in surface epithelia. For IFs comprised of keratins 5 and 14 (K5, K14), found in basal keratinocytes of epidermis, bundling can be self-driven through interactions between K14's carboxy-terminal tail domain and two regions in the central α-helical rod domain of K5. Here, we exploit theoretical principles and computational modeling to investigate how such cis-acting determinants best promote IF crosslinking. We develop a simple model where keratin IFs are treated as rigid rods to apply Brownian dynamics simulation. Our findings suggest that long-range interactions between IFs are required to initiate the formation of bundlelike configurations, while tail domain-mediated binding events act to stabilize them. Our model explains the differences observed in the mechanical properties of wild-type versus disease-causing, defective IF networks. This effort extends the notion that the structural support function of keratin IFs necessitates a combination of intrinsic and extrinsic determinants, and makes specific predictions about the mechanisms involved in the formation of crosslinked keratin networks in vivo.     

Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a coil2 fragment of human lamin A

The elementary building block of all intermediate filaments (IFs) is a dimer featuring a central α-helical rod domain flanked by the N- and C-terminal end domains. In nuclear IF proteins (lamins), the rod domain consists of two coiled-coil segments, coil1 and coil2, that are connected by a short non-helical linker. Coil1 and the C-terminal part of coil2 contain the two highly conserved IF consensus motifs involved in the longitudinal assembly of dimers. The previously solved crystal structure of a lamin A fragment (residues 305-387) corresponding to the second half of coil2 has yielded a parallel left-handed coiled coil. Here, we present the crystal structure and solution properties of another human lamin A fragment (residues 328-398), which is largely overlapping with fragment 305-387 but harbors a short segment of the tail domain. Unexpectedly, no parallel coiled coil forms within the crystal. Instead, the α-helices are arranged such that two anti-parallel coiled-coil interfaces are formed. The most significant interface has a right-handed geometry, which is accounted for by a characteristic 15-residue repeat pattern that overlays with the canonical heptad repeat pattern. The second interface is a left-handed anti-parallel coiled coil based on the predicted heptad repeat pattern. In solution, the fragment reveals only a weak dimerization propensity. We speculate that the C-terminus of coil2 might unzip, thereby allowing for a right-handed coiled-coil interface to form between two laterally aligned dimers. Such an interface might co-exist with a heterotetrameric left-handed coiled-coil assembly, which is expected to be responsible for the longitudinal A_CN contact.