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ScienceDirect Intermediate filament structure: the bottom-up approach Anastasia A Chernyatina, Dmytro Guzenko and Sergei V Strelkov Intermediate filaments (IFs) result from a key cytoskeletal protein class in metazoan cells, but currently there is no consensus as to their three-dimensional architecture. IF proteins form elongated dimers based on the coiled-coil structure within their central ‘rod’ domain. Here we focus on the atomic structure of this elementary dimer, elucidated using Xray crystallography on multiple fragments and electron paramagnetic resonance experiments on spin-labelled vimentin samples. In line with conserved sequence features, the rod of all IF proteins is composed of three coiled-coil segments containing heptad and hendecad repeats and interconnected by linkers. In addition, the next assembly intermediate beyond the dimer, the tetramer, could be modelled. The impact of these structural results towards understanding the assembly mechanism is discussed.

organization. In their primary structure, one can discern a highly a-helical central domain (‘rod’), flanked by the N-terminal (‘head’) and C-terminal (‘tail’) domains highly variable in sequence and length and typically containing little secondary structure [3]. The rod domain forms an a-helical coiled coil, one of the principal motifs of protein architecture (Box 1), composed of two parallel in register chains. Most IF proteins form homodimers, although preferential heterodimers such as keratins as well as further heterotypic assemblies in vivo are also observed [4]. It is the coiled-coil rod that is responsible for the overall elongated shape of the elementary dimer, which measures 45–48 nm in cytoplasmic IF proteins and 50–52 nm in nuclear lamins [5,6].

Addresses Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Belgium

Even though the assembly of IFs in their natural habitat — either the cytoplasm or the nucleus — occurs in the presence of many other protein factors, various IF types could also be reproduced in vitro by manipulating with solution parameters. Thus IFs qualify as self-assembling systems [7]. There are several assembly principles that are valid for all IFs. First, the axes of the dimers are aligned roughly in the filament direction; second, dimers associate laterally via several distinct modes [8]; and finally, longitudinal growth of the filament depends on the end-to-end interaction of dimers. The exact assembly pathway varies for different IF types [4,9]. For instance, vimentin is found in a low molarity neutral buffer in the form of tetramers resulting from two half-staggered antiparallel dimers (so-called A11 tetramers). Subsequent increase of ionic strength yields higher lateral assembly intermediates such as octamers and the so-called unitlength filaments (ULFs) typically containing 4 octamers, followed by longitudinal assembly that eventually results in native-like filaments [4,10].

Corresponding author: Strelkov, Sergei V ([email protected])

Current Opinion in Cell Biology 2015, 32:65–72 This review comes from a themed issue on Cell architecture Edited by Sandrine Etienne-Manneville and Elly M Hol

http://dx.doi.org/10.1016/j.ceb.2014.12.007 0955-0674/# 2015 Elsevier Ltd. All rights reserved.

Introduction Intermediate filaments (IFs) have a clear-cut importance for the functioning of metazoan cells. However, at present there is no consensus with regard to the architecture and assembly mechanism of these nanofilaments. In fact, the 10 nm wide IFs have proven to be much more challenging for structural studies than microtubules and F-actin, for both of which a complete 3D description, including atomic resolution data on their respective building blocks and the symmetry of the assembled filament, has long been available [1,2]. All IF proteins, classified into five main families (‘types’, see http://www.interfil.org), have a distinct tripartite www.sciencedirect.com

Here we will focus on the considerable advances that have been made in the recent years towards the atomic structure of the elementary IF dimer, contributing to a better understanding of both the IF architecture and assembly mechanism.

Sequence signature of IF proteins Early on, it was realized that the seven-residue (‘heptad’) periodicity in the distribution of hydrophobic residues, indicative of a left-handed coiled coil (Box 1), is the prevalent motif of the IF rod. Correspondingly, algorithms looking specifically for regions with heptad repeats Current Opinion in Cell Biology 2015, 32:65–72

66 Cell architecture

Box 1 Structural principles of a-helical coiled coils. A classical left-handed coiled coil can be recognized by a heptad (seven-residue) periodic pattern HxxHxxx, where H stands for mainly hydrophobic residues such as Leu, Ile, Val or Met. By convention, this periodicity is designated (abcdefg)n, with hydrophobic positions a and d. If such a sequence forms an a-helix (which contains about 3.6 residues per turn, meaning that a heptad roughly corresponds to two turns), residues in a and d positions will locate approximately on one side of the helix. This promotes ‘zipping’ together of two or more a-helices, which results in a left-handed supercoil with a common hydrophobic core [59]. Another, less common hydrophobic pattern is a hendecad (11 residues/3 turns), designated (abcdefghijk)n, with the core made by positions a, d and h (sometimes position e is also involved) [60]. This results in a nearly parallel (slightly right-handed) bundle of a-helices. For additional detail see Figure 2d in [61]. A discontinuity in a heptad pattern equivalent to a four-residue insert is called a ‘stutter’. As shown experimentally, this discontinuity can be tolerated within a continuous heptad-based left-handed coiled coil, but causes its local unwinding [62]. Indeed, a heptad extended by four residues is equivalent to a single instance of a hendecad. We also note that a discontinuity equivalent to one-residue insert in a regular heptad pattern can be seen as two consecutive stutters.

were employed, which resulted in the original model of the IF dimer (reviewed in [9]) that included four segments of continuous left-handed coiled coil (with the last segment containing a ‘stutter’, see Box 1). The connections between the segments were assumed to be made by non-a-helical linkers. This model of rod organization, although merely a result of a particular sequence analysis algorithm, regretfully, still appears ‘as is’ in many current IF-related publications. In fact, subsequent research has introduced substantial corrections to this model. Figure 1 shows the aligned rod domain sequences of the representatives of four important IF types, human vimentin (type III chain), keratin K5 (type I), keratin K14 (type II) and lamin A (type V). As can be seen, secondary structure prediction algorithms such as Jpred3 [11] suggest only three continuous ahelical segments (coil1A, coil1B and coil2) for all four sequences. Interestingly, early predictions made soon after the first IF protein sequences had been obtained also suggested three a-helical segments [12]. In addition, the solvent exposure of residues along each sequence can be estimated using the NetSurfP predictor [13]. Logically, a coiled-coil sequence should (a) have high a-helical propensity and (b) display a periodic pattern of buried residues that form its hydrophobic core. For the larger part of the IF rod, this pattern does correspond to heptads (Figure 1). In addition, in several places but most importantly at the beginning of coil2, a different periodicity is observed, namely an 11-residue (hendecad) repeat (see Box 1). While the overall sequence conservation across different IF types is rather low, it is the ‘signature’ features of the rod domain that define an IF protein [14]. In particular, Current Opinion in Cell Biology 2015, 32:65–72

both the length of each of the three coiled-coil segments and their specific heptad/hendecad patterns are conserved in nearly all IF types (Figure 1). Coil1A contains close to 42 residues (6 heptad repeats). Coil1B is typically close to 102 residues (13 heptads and one hendecad), but includes 42 additional residues (6 heptads) in nuclear lamins. Finally, coil2 is close to 140 residues (13 heptads and 4 hendecads). Furthermore, all IF proteins contain two conserved ‘consensus’ motifs at either termini of the rod domain, namely in the middle part of coil1A and at the very end of coil2 [15]. These regions appear to be of special importance for the filament assembly [4].

Experimental studies of IF dimer Over the last years, considerable efforts have been made towards the experimental elaboration of the IF dimer structure. Principally, two methods, X-ray crystallography and electron paramagnetic resonance on site-directed spin-labelled protein samples (SDSL-EPR) were employed. With few exceptions, the experiments have been carried out with human vimentin as the ‘model’ IF protein. Crystallization of a full-length IF protein is clearly an impossible task, due to its intrinsic propensity to assemble into filaments. To overcome this, we originally proposed a ‘divide-and-conquer’ strategy which relied on crystallization of short fragments [16]. All crystallographic data on IF proteins obtained thus far by several laboratories result from this approach. Since the success of protein crystallization cannot be predicted at present, many dozens of fragments with varying ends had to be recombinantly prepared and screened using standard methods. In our estimate, only 15% of these fragments yielded crystals suitable for X-ray studies. Figure 2a and b show an overview of atomic structures resolved to date for the rod domains of vimentin [15,17,18,19,20,21], keratin 5/14 heterodimer [22] and lamins A and B1 [23,24], as well as the immunoglobulin fold present within the lamin tail domain [24,25]. In addition, structures of lamin A fragments carrying disease-related mutations in coil2 and the immunoglobulin fold have been determined [26,27]. Altogether the resolved fragments cover close to 100% of the rod domain except for the L12 region. The lengths of the crystallized fragments range between 37 and 106 residues. A general question with regard to the ‘divide-and-conquer’ strategy is whether the relatively short fragments fold into the correct native structure. Most of the crystallized fragments did yield the expected parallel coiled-coil dimers (Figure 2a), although in several cases aberrant structures were obtained (briefly discussed below) [18,21]. Furthermore, many of the resolved fragments are overlapping, and they generally show a good match of the 3D coordinates, as seen in Figure 2c and D (some long-range deviations are not unexpected, due to the www.sciencedirect.com

Intermediate filament structure Chernyatina, Guzenko and Strelkov 67

Figure 1

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Primary structure of the IF rod domain. Amino acid sequences for human vimentin (numbered above the sequence), keratins 5 and 14, and lamin A are aligned. Secondary structure prediction using the Jpred3 algorithm [11] is shown for each sequence (wave underline indicates a-helix, simple underline indicates b-strand). Residues predicted by the NetSurfP algorithm [13] to be buried in the hydrophobic core are highlighted in yellow. Residues that give a typical ‘core-like’ signal in SDSL-EPR experiments [34] are highlighted in blue. Residues for which both indications agree are highlighted in green. Heptad and hendecad assignments are indicated, with the latter also highlighted in violet. The hendecad repeat in the middle of coil2 is traditionally labelled as ‘stutter’. Proline residues are highlighted in grey.

flexibility of the extended coiled-coil molecules). Moreover, the structure of the second half of coil2 has been obtained not only for vimentin but also for keratin 5/14 heterodimer, lamin A and lamin B1, revealing an excellent agreement of all structures (Figure 2b). The SDSL-EPR experiments on vimentin samples have been a systematic effort of John Hess and colleagues [19,28–35,36]. These experiments probe the properties of one residue at a time in the context of the full-length protein. The residue is first mutated to cysteine, while the single native Cys328 of vimentin is replaced by an alanine, and then a spin label is chemically attached. Thereafter the collection of the EPR spectrum can provide semi-quantitative information about the structural rigidity of the environment of the label. In addition, the proximity of labels residing in different chains can be detected [34]. In particular, this method allows identification of the residues located in the core positions of the www.sciencedirect.com

(homo)dimeric coiled coil. All such residues established to date in the vimentin rod are marked in Figure 1. Importantly, throughout the rod domain, there is a nearly perfect match between the core positions indicated by sequence analysis, SDSL-EPR experiments, and crystal structures. This also applies to the regions with hendecads rather than heptads.

Coil1 structure The originally determined crystal structure of a vimentin fragment corresponding to coil1A showed a monomer rather than a coiled-coil dimer [15], which was related to the low thermodynamic stability of this segment. Introducing an artificial mutation Y117L into this fragment resulted in a coiled coil with increased stability, which could also be resolved as such (PDB code 3G1E, Figure 2a) [17]. The hydrophobic core of the dimer followed the predicted heptad pattern (Figure 1), which was also confirmed using SDSL-EPR for the full-length protein [32]. In addition, structures of two similar fragments each Current Opinion in Cell Biology 2015, 32:65–72

68 Cell architecture

Figure 2

(a) Coil1 fragments

(b) Coil2 fragments

1GK6 (385-412)

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Current Opinion in Cell Biology

Structural data on the IF dimer. (a) Crystal structures of all coil1 fragments available to date, shown as ribbon diagrams. The fragments, all coming from human vimentin, are labelled by their PDB codes, with the residue range indicated in brackets [15,17,19,21]. The fragments are oriented in the same way. The artificial mutation Y117L is marked with a red dot. (b) Crystal structures of coil2 fragments of vimentin, the keratin 5/14 heterodimer and lamin A; the structure of the immunoglobulin fold present in the tail domain of lamin A (1IFR) is also shown [25]. The 1GK6 structure contains a leucine zipper domain (grey) fused to the vimentin fragment. Artificial disulfide bridge in the 3TRT fragment is marked with a green dot. The available structures of the coil2 and tail fragments of lamin B1 ([24], see also www.pdb.org) are highly similar to the corresponding fragments of lamin A and not shown. (c) Least squares superposition of all available coil1 fragments. (d) Least-squares superposition of coil2 fragments. (e) Atomic model of a complete vimentin dimer. The model, assuming perfect twofold symmetry, was produced by computational modelling using all available crystal structures of fragments as templates (Guzenko et al., in preparation). The regions of the rod for which no experimental structures are available and which were modelled ab initio are shown in grey. The poorly ordered head and tail domains (semitransparent red and blue respectively) are best given by a cloud-like representation (40 modelled conformations). (f) Atomic model of vimentin tetramer. The alignment of two dimers was based on the 3UF1 structure. The twofold axis of the tetramer is indicated.

corresponding to the almost complete coil1B were determined (PDB codes 3SWK and 3UF1) [19,21], confirming a regular left-handed coiled-coil structure encompassing nearly the entire length of coil1B but also indicating a single instance of a hendecad repeat close to its C-terminus, in line with predictions (Figures 1 and 2a). Although poorly conserved, the linker L1 is consistently revealed as a distinct drop in the predicted a-helicity in various IF types (Figure 1); in several cases, its sequence includes proline residues which are known to disrupt or cap the ends of a-helices. Moreover, due to varying lengths of the linker, the heptad phase shift from coil1A to coil1B differs across IF proteins. In vimentin, keratins 5 and 14 and lamin A this phase shift corresponds to Current Opinion in Cell Biology 2015, 32:65–72

five-residue, two-residue, one-residue and one-residue inserts, respectively. Only the last case is, in principle, compatible with a continuous coiled coil from coil1A into 1B, with two stutters and, correspondingly, local coiled-coil unwinding at the linker (Box 1). A fragment including coil1A, linker L1 and part of coil1B could also be crystallized (PDB code 3SSU) [21]. However, coil1A was found to be completely disordered in these crystals, and it was therefore unclear whether this segment formed a coiled coil. Subsequently, the latter fragment was modified to carry the stabilizing Y117L mutation, but surprisingly the resulting crystal structure (3S4R) [21] still did not show a coiled-coil dimer for coil1A, as had been observed in the 3G1E structure. www.sciencedirect.com

Intermediate filament structure Chernyatina, Guzenko and Strelkov 69

Instead, a single continuous a-helix was observed spanning coil1A, linker L1 and coil1B. While the 1B part formed the predicted coiled-coil dimer, the two 1A ahelices splayed apart (Figure 2a) and were involved in contacts with neighbouring chains in the crystal lattice. The accumulated, rather complex data on linker L1 and the adjacent regions allows two distinct interpretations. The first model, which we still consider as the leading hypothesis, assumes that the 1A segment does form a coiled coil in the full dimer context, followed by a discrete non-helical linker L1. Based on exhaustive SDSL-EPR mapping, it was proposed that coil1A of vimentin should extend up to residue G142 while coil1B should start at residue G147 already, and that the linker, corresponding to residues 143–146, should have a well-ordered structure rather than being flexible [19]. Indeed, the presence of glycine residues at either end would allow abrupt deviation from the a-helical geometry necessary to compensate the heptad phase shift across the linker. However, the non-helical conformation of L1 has escaped direct visualization thus far. The alternative model of the coil1A-L1 conformation assumes that the weak intradimeric coiled coil of 1A is not formed, but the individual 1A helices are kept away from each other by a fully a-helical structure of linker L1, just like observed in the 3S4R crystals (Figure 2c) [21]. Support to this model comes from the hypothesis that the N-terminal ‘consensus’ region of the rod should be involved in the longitudinal dimer–dimer contact within the assembled filament. Indeed, with a distinct hydrophobic patch of core residues on one side, the monomeric coil1A could be ‘reactive’ towards the C-terminal part of the rod from another dimer [21]. Finally, it has been speculated that both ‘zipped’ and ‘unzipped’ conformations of coil1A might exist at different IF assembly stages [17,37].

Coil2 structure A small C-terminal portion of vimentin coil2 was first crystallized as a fusion with GCN4 leucine zipper. This was followed by a crystal structure of the C-terminal half of coil2 (Figure 2b) [15]. The latter structure confirmed, in particular, the unwinding of the left-handed coiled coil at the predicted stutter (residue 351). More of a surprise, the crystal structure of a vimentin fragment corresponding to the first half of coil2 (PDB code 3KLT) showed an antiparallel four-helix bundle formed by the N-terminal parts of the fragment [18]. At the same time, the core of this bundle was made by residues following a hendecad pattern, just as predicted from the sequence (Figure 1). Subsequently we have also crystallized the same fragment with a single point mutation to Cys at its N-terminus, that is, first predicted core position 265 [20]. This mutation forced the correct dimerization of the N-terminal part of coil2 via a disulfide link (PDB code 3TRT, Figure 2b). This structure confirms that coil2 starts with a www.sciencedirect.com

hendecad-based parallel bundle that extends all the way through and slightly beyond the parts known before as coil2A and L2, followed by a regular heptad-based coiled coil. These crystallographic data are in excellent agreement with the core positions suggested by the SDSLEPR scanning of residues 281 through 337 (Figure 1) [28,29].

Linker L12, head and tail domains The linker L12 is the only part of the rod still refractory to crystallographic studies. It varies only modestly in length (about 15 residues) across different IF types, and also appears to contain a conserved pattern HHH (H — hydrophobic and x — any residue) often predicted as a short b-strand (Figure 1). The linker L12 appears to be a flexible hinge connecting the fairly rigid coil1B and coil2 segments, which is qualitatively supported by electron microscopy (EM) on rotary-shadowed isolated vimentin rods which often show a kink in the middle [10]. Both head and tail domains appear to be intrinsically disordered in most IF proteins [38], the known exception being a small part of the lamin tail that forms an immunoglobulin fold [25]. The lack of structural order in these two terminal regions, at least at lower assembly stages, makes it difficult to establish good links to their functional roles. At the same time, it is known that the vimentin head domain is essential for filament assembly, a process regulated by phosphorylation within this region [39]. In the full-length vimentin dimer, the head domain seems to fold back on coil1A, as SDSL-EPR data indicated an interaction of residue 17 of the head domain and residue 137 located near the C-end of coil1A [32,33], while chemical crosslinking suggested that the head domain could come in proximity of coil1A and even the beginning of coil1B [4]. In addition, recent EPR analyses suggest that the proximal part of the vimentin tail forms a relatively ordered dimeric structure up to residue 420, while the distal part of the tail is highly flexible but does become more ordered in the course of filament assembly [36]. However, due to high sequence variability one cannot readily extrapolate these results to the head and tail domains of other IFs.

Structure of IF tetramer Interestingly, the crystals of the coil1B vimentin fragment with PDB code 3UF1 revealed tetramers consisting of two dimers running antiparallel [19]. This arrangement reproduces the A11-type tetramer of the full-length protein. Indeed, the observed 3UF1 tetramer is centred on residue 191 just like the full-length tetramers, as shown using SDSL-EPR and compatible with early chemical crosslinking data [8,35]. The formation of the 3UF1 tetramer involves specific interdimeric salt bridges and is accompanied by some distortion in each dimer, so that they lose their axial twofold symmetry [19]. As a result, ‘endless’ lateral association of the dimers via the A11 Current Opinion in Cell Biology 2015, 32:65–72

70 Cell architecture

mode is suppressed. The 3UF1 structure can be used as a template to build a realistic atomic model of the compete vimentin tetramer (Figure 2f). The length of the latter, including the modelled terminal domains, is 62 nm, which is in good agreement with the length of the negatively stained ULFs (about 60 nm) [40].

biophysical techniques. In particular, kinetics of vimentin assembly can be studied based on EM and scanning force microscopy data [49,50]. In addition, small angle X-ray scattering [51,52,53] and cryo EM-based tomography [54,55] can contribute low-resolution 3D structure information on higher assembly intermediates and mature IFs.

Coiled-coil stability

While the ‘bottom-up’ approach to the vimentin structure outlined here resulted in significant advancements, the large differences observed across various IF types suggest that a single ‘unifying’ model of IF assembly is rather unlikely. Indeed, beyond the IF ‘signature’ features, the rod domains of different IFs are highly variable at the sequence level, and more so are the functional terminal domains [9]. Furthermore, one should expect different IF types to be more deviant in their architecture and hence properties than the structures of their constitutive dimers, due to a cumulative effect of small differences towards higher assembly levels. In particular, nuclear lamins seem to assemble via a radically different pathway compared to cytoplasmic IF proteins [4]. Even for vimentin and desmin, two closely related type III proteins capable of coassembly, distinct mechanical properties of the in vitro assembled filaments were observed [56]. At the other extreme, two ‘orphan’ IFs from the eye lens, with such unusual features as the reduced coil2 domain in BFSP1 and severely altered N-terminal ‘consensus motif’ of the IF rod in BFSP2 [57], or equally the newly identified insect cytoplasmic IF protein isomin [14,58], are almost certain to assemble in unique, yet to be explored ways.

A ‘by-product’ of preparing IF rod fragments for crystallography was the observation that their dimerization capacity is highly variable. The full-length vimentin molecule is dimeric in as high as 6 M urea [10], which suggests that the complete coiled-coil rod, being a cooperative system, is very stable. At the same time, the coiled coil formed by a vimentin fragment corresponding to the relatively short coil1A showed only a marginal stability, having a melting temperature of 32 8C at 1 mg/ml [17]. Moreover, while an artificial mutation Y117L had a stabilizing effect on this coiled-coil segment, introducing this mutation into the full-length protein resulted in aberrant assembly, largely confined to the ULF stage [17]. Likewise, a vimentin fragment corresponding to the N-terminal half of coil2 and containing hendecad repeats was monomeric in solution [18]. Thus, ‘unzipping’ of some parts of the rod might be important for normal IF assembly and requires further investigation.

Conclusions and outlook Redundant crystallographic data on vimentin fragments, supported by sequence-based predictions and SDSLEPR experiments, have provided a nearly complete picture of the IF rod structure, which is composed of three ahelical segments interconnected by linkers. The first two segments contain almost exclusively heptad repeats, yielding a regular left-handed coiled coil, but the Nterminal region of the third segment features hendecad periodicity that results in a parallel a-helical bundle. Potentially, this atomic resolution information opens a perspective of modelling the IF assembly mechanism in three dimensions. However, such modelling is not only highly demanding computationally, but it is also complicated by the observed fragility of the IF architecture. The latter property is evident from the studies of diseaserelated, mostly single point mutations in IF proteins that frequently lead to catastrophic consequences up to the complete loss of assembly [41,42,43,44]. This means that even small inaccuracies in the dimer or tetramer model are likely to undermine the validity of subsequent modelling. This stresses the importance of collecting further experimental data on higher levels of IF architecture, beyond the classical crosslinking experiments [8], as these data can provide the necessary restraints for the computational modelling. Such data can be obtained on the basis in vitro assembly studies, such as the ones recently performed on keratins [45,46] and lamins [47,48], using a multitude of Current Opinion in Cell Biology 2015, 32:65–72

Acknowledgements The authors are grateful to Harald Herrmann for many exciting discussions, and to Stephen Weeks for helpful feedback on the manuscript. This research was supported by the KU Leuven (grant OT13/097 to SVS), by Research Foundation Flanders FWO (grant G070912N to SVS) and by the EU COST Action ‘Nanonet’.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Nogales E, Wolf SG, Downing KH: Structure of the alpha beta tubulin dimer by electron crystallography. Nature 1998, 391:199-203.

2.

Holmes KC, Popp D, Gebhard W, Kabsch W: Atomic model of the actin filament. Nature 1990, 347:44-49.

3.

Parry DA, Strelkov SV, Burkhard P, Aebi U, Herrmann H: Towards a molecular description of intermediate filament structure and assembly. Exp Cell Res 2007, 313:2204-2216.

4.

Herrmann H, Aebi U: Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds. Annu Rev Biochem 2004, 73:749-789.

5.

Aebi U, Cohn J, Buhle L, Gerace L: The nuclear lamina is a meshwork of intermediate-type filaments. Nature 1986, 323:560-564. www.sciencedirect.com

Intermediate filament structure Chernyatina, Guzenko and Strelkov 71

6.

Quinlan RA, Hatzfeld M, Franke WW, Lustig A, Schulthess T, Engel J: Characterization of dimer subunits of intermediate filament proteins. J Mol Biol 1986, 192:337-349.

7.

Herrmann H, Bar H, Kreplak L, Strelkov SV, Aebi U: Intermediate filaments: from cell architecture to nanomechanics. Nat Rev Mol Cell Biol 2007, 8:562-573.

8.

Steinert PM, Marekov LN, Parry DA: Diversity of intermediate filament structure. Evidence that the alignment of coiled-coil molecules in vimentin is different from that in keratin intermediate filaments. J Biol Chem 1993, 268:24916-24925.

9.

Parry DAD, Steinert PM: Intermediate filaments: molecular architecture, assembly, dynamics and polymorphism. Q Rev Biophys 1999, 32:99-187.

10. Herrmann H, Haner M, Brettel M, Muller SA, Goldie KN, Fedtke B, Lustig A, Franke WW, Aebi U: Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains. J Mol Biol 1996, 264:933-953. 11. Cole C, Barber JD, Barton GJ: The Jpred 3 secondary structure prediction server. Nucleic Acids Res 2008, 36:W197-W201. 12. Quax-Jeuken YE, Quax WJ, Bloemendal H: Primary and secondary structure of hamster vimentin predicted from the nucleotide sequence. Proc Natl Acad Sci U S A 1983, 80:35483552. 13. Petersen B, Petersen TN, Andersen P, Nielsen M, Lundegaard C: A generic method for assignment of reliability scores applied to solvent accessibility predictions. BMC Struct Biol 2009, 9:51. 14. Herrmann H, Strelkov SV: History and phylogeny of  intermediate filaments: now in insects. BMC Biol 2011, 9:1-5. Common sequence and structural features that define the ‘signature’ of an IF protein are discussed, in the context of a recent discovery of an insect cytoplasmic IF protein, isomin. 15. Strelkov SV, Herrmann H, Geisler N, Wedig T, Zimbelmann R, Aebi U, Burkhard P: Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly. Embo J 2002, 21:1255-1266. 16. Strelkov SV, Herrmann H, Geisler N, Lustig A, Ivaninskii S, Zimbelmann R, Burkhard P, Aebi U: Divide-and-conquer crystallographic approach towards an atomic structure of intermediate filaments. J Mol Biol 2001, 306:773-781. 17. Meier M, Padilla GP, Herrmann H, Wedig T, Hergt M, Patel TR, Stetefeld J, Aebi U, Burkhard P: Vimentin coil 1A-A molecular switch involved in the initiation of filament elongation. J Mol Biol 2009, 390:245-261. 18. Nicolet S, Herrmann H, Aebi U, Strelkov SV: Atomic structure of vimentin coil 2. J Struct Biol 2010, 170:369-376. 19. Aziz A, Hess JF, Budamagunta MS, Voss JC, Kuzin AP, Huang YJ,  Xiao R, Montelione GT, Fitzgerald PG, Hunt JF: The structure of vimentin linker 1 and rod 1B domains characterized by sitedirected spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography. J Biol Chem 2012, 287:28349-28361. Using a combination of X-ray crystallography and SDSL-EPR, the authors obtain structural insight into the first half of vimentin’s rod domain. Most interestingly, the crystal structure of a fragment containing full coil1B revealed tetramers, centred on residue 191, which correspond to the arrangement in the A11-type tetramers made by the full-length protein. In addition, the EPR measurements suggest that the linker L1 has a rigid structure. 20. Chernyatina AA, Strelkov SV: Stabilization of vimentin coil2  fragment via an engineered disulfide. J Struct Biol 2012, 177:46-53. By solving the X-ray structure of a disulfide-stabilized vimentin fragment that corresponds to the beginning of coil2, direct evidence on the hendecad-based parallel a-helical structure in this region is obtained. 21. Chernyatina AA, Nicolet S, Aebi U, Herrmann H, Strelkov SV:  Atomic structure of the vimentin central alpha-helical domain and its implications for intermediate filament assembly. Proc Natl Acad Sci U S A 2012, 109:13620-13625. This paper presents three novel crystal structures of vimentin fragments that span coil1A, linker L1 and coil1B. By taking together all structural data obtained to date, generation of a 3D model of the complete IF rod domain becomes possible. The implications of this model towards IF assembly mechanism are discussed. www.sciencedirect.com

22. Lee CH, Kim MS, Chung BM, Leahy DJ, Coulombe PA: Structural basis for heteromeric assembly and perinuclear organization  of keratin filaments. Nat Struct Mol Biol 2012, 19:707-715. The first crystal structure of a cytokeratin rod fragment is reported. The structure reveals the heterodimer of K5 and K14 fragments that form the major part of coil2, confirming the structural organization known already from studies of vimentin and lamins. In the crystals, adjacent dimers are interconnected by disulfide bonds, which correlates with the role of such bonds in keratin IFs. 23. Strelkov SV, Schumacher J, Burkhard P, Aebi U, Herrmann H: Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins. J Mol Biol 2004, 343:1067-1080. 24. Ruan J, Xu C, Bian C, Lam R, Wang JP, Kania J, Min J, Zang J: Crystal structures of the coil 2B fragment and the globular tail domain of human lamin B1. FEBS Lett 2012, 586:314-318. 25. Dhe-Paganon S, Werner ED, Chi YI, Shoelson SE: Structure of the globular tail of nuclear lamin. J Biol Chem 2002, 277:17381-17384. 26. Magracheva E, Kozlov S, Stewart CL, Wlodawer A, Zdanov A: Structure of the lamin A/C R482W mutant responsible for dominant familial partial lipodystrophy (FPLD). Acta Crystallogr Sect F Struct Biol Cryst Commun 2009, 65:665-670. 27. Bollati M, Barbiroli A, Favalli V, Arbustini E, Charron P, Bolognesi M: Structures of the lamin A/C R335W and E347K mutants: implications for dilated cardiolaminopathies. Biochem Biophys Res Commun 2012, 418:217-221. 28. Hess JF, Voss JV, FitzGerald PG: Real-time observation of coiled-coil domains and subunit assembly in intermediate filaments. J Biol Chem 2002, 277:35516-35522. 29. Hess JF, Budamagunta MS, Shipman RL, FitzGerald PG, Voss JC: Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance. Biochemistry 2006, 45:11737-11743. 30. Hess JF, Budamagunta MS, FitzGerald PG, Voss JC: Characterization of structural changes in vimentin bearing an epidermolysis bullosa simplex-like mutation using sitedirected spin labeling and electron paramagnetic resonance. J Biol Chem 2005, 280:2141-2146. 31. Pittenger JT, Hess JF, Budamagunta MS, Voss JC, Fitzgerald PG: Identification of phosphorylation-induced changes in vimentin intermediate filaments by site-directed spin labeling and electron paramagnetic resonance. Biochemistry 2008, 47:10863-10870. 32. Aziz A, Hess JF, Budamagunta MS, Voss JC, Fitzgerald PG: Sitedirected spin labeling and electron paramagnetic resonance determination of vimentin head domain structure. J Biol Chem 2010, 285:15278-15285. 33. Aziz A, Hess JF, Budamagunta MS, FitzGerald PG, Voss JC: Head and rod 1 interactions in vimentin: identification of contact sites, structure, and changes with phosphorylation using sitedirected spin labeling and electron paramagnetic resonance. J Biol Chem 2009, 284:7330-7338. 34. Budamagunta M, Hess J, Fitzgerald P, Voss J: Describing the structure and assembly of protein filaments by EPR spectroscopy of spin-labeled side chains. Cell Biochem Biophys 2007, 48:45-53. 35. Hess JF, Budamagunta MS, Voss JC, FitzGerald PG: Structural characterization of human vimentin rod 1 and the sequencing of assembly steps in intermediate filament formation in vitro using site-directed spin labeling and electron paramagnetic resonance. J Biol Chem 2004, 279:44841-44846. 36. Hess JF, Budamagunta MS, Aziz A, FitzGerald PG, Voss JC:  Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly. Protein Sci 2013, 22:47-55. The presented SDSL-EPR data provide a first glance at the structure of the vimentin tail domain. The region up to residue 420 appears more ordered, with chains running in parallel, although inconsistent with a coiled-coil structure. The remainder of the tail domain is largely disordered at early assembly stages, but becomes more rigid in mature IFs. Current Opinion in Cell Biology 2015, 32:65–72

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37. Smith TA, Strelkov SV, Burkhard P, Aebi U, Parry DA: Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1. J Struct Biol 2002, 137:128-145.

49. Kirmse R, Portet S, Mucke N, Aebi U, Herrmann H, Langowski J: A quantitative kinetic model for the in vitro assembly of intermediate filaments from tetrameric vimentin. J Biol Chem 2007, 282:18563-18572.

38. Guharoy M, Szabo B, Contreras Martos S, Kosol S, Tompa P: Intrinsic structural disorder in cytoskeletal proteins. Cytoskeleton (Hoboken) 2013, 70:550-571.

50. Portet S: Dynamics of in vitro intermediate filament length distributions. J Theor Biol 2013, 332:20-29.

39. Eriksson JE, He T, Trejo-Skalli AV, Harmala-Brasken AS, Hellman J, Chou YH, Goldman RD: Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments. J Cell Sci 2004, 117:919-932.

51. Sokolova AV, Kreplak L, Wedig T, Mucke N, Svergun DI, Herrmann H, Aebi U, Strelkov SV: Monitoring intermediate filament assembly by small-angle X-ray scattering reveals the molecular architecture of assembly intermediates. Proc Natl Acad Sci U S A 2006, 103:16206-16211.

40. Herrmann H, Haner M, Brettel M, Ku NO, Aebi U: Characterization of distinct early assembly units of different intermediate filament proteins. J Mol Biol 1999, 286:1403-1420. 41. Szeverenyi I, Cassidy AJ, Chung CW, Lee BT, Common JE, Ogg SC, Chen H, Sim SY, Goh WL, Ng KW et al.: The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases. Hum Mutat 2008, 29:351-360. 42. Clemen CS, Herrmann H, Strelkov SV, Schroder R:  Desminopathies: pathology and mechanisms. Acta Neuropathol 2013, 125:47-75. This review discusses the numerous mutations in IF protein desmin that were associated with (cardio)myopathies. The detrimental effect of these mutations has been studied at various levels, from in vitro assembly experiments with recombinant desmin to cell culture and clinical pathophysiological data.

52. Brennich ME, Bauch S, Vainio U, Wedig T, Herrmann H, Koster S:  Impact of ion valency on the assembly of vimentin studied by quantitative small angle X-ray scattering. Soft Matter 2014, 10:2059-2068. In this study, SAXS was employed to systematically study the assembly of IFs by controlling the solution environment. 53. Brennich ME, Nolting JF, Dammann C, Noding B, Bauch S, Herrmann H, Pfohl T, Koster S: Dynamics of intermediate filament assembly followed in micro-flow by small angle X-ray scattering. Lab Chip 2011, 11:708-716. 54. Goldie KN, Wedig T, Mitra AK, Aebi U, Herrmann H, Hoenger A: Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography. J Struct Biol 2007, 158:378-385.

43. Herrmann H, Strelkov SV, Burkhard P, Aebi U: Intermediate filaments: primary determinants of cell architecture and plasticity. J Clin Invest 2009, 119:1772-1783.

55. Kirmse R, Bouchet-Marquis C, Page C, Hoenger A: Threedimensional cryo-electron microscopy on intermediate filaments. Methods Cell Biol 2010, 96:565-589.

44. Gangemi F, Degano M: Disease-associated mutations in the coil 2B domain of human lamin A/C affect structural properties that mediate dimerization and intermediate filament formation. J Struct Biol 2013, 181:17-28.

56. Schopferer M, Bar H, Hochstein B, Sharma S, Mucke N, Herrmann H, Willenbacher N: Desmin and vimentin intermediate filament networks: their viscoelastic properties investigated by mechanical rheometry. J Mol Biol 2009, 388:133-143.

45. Lichtenstern T, Mucke N, Aebi U, Mauermann M, Herrmann H:  Complex formation and kinetics of filament assembly exhibited by the simple epithelial keratins K8 and K18. J Struct Biol 2012, 177:54-62. Assembly of keratin 8/18 filaments was studied in detail using an in vitro assembly protocol. The kinetics of assembly, while also proceeding via the ULF stage, is much faster than that of the previously examined vimentin filaments. 46. Kayser J, Grabmayr H, Harasim M, Herrmann H, Bausch AR: Assembly kinetics determine the structure of keratin networks. Soft Matter 2012, 8:8873-8879. 47. Ben-Harush K, Wiesel N, Frenkiel-Krispin D, Moeller D, Soreq E, Aebi U, Herrmann H, Gruenbaum Y, Medalia O: The supramolecular organization of the C. elegans nuclear lamin filament. J Mol Biol 2009, 386:1392-1402. 48. Zwerger M, Medalia O: From lamins to lamina: a structural  perspective. Histochem Cell Biol 2013, 140:3-12. This review presents the current understanding of the nuclear lamina from the structural point of view. The authors suggest that the latest developments in the imaging techniques such as cryo-EM can help further progress in this field.

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57. Song SH, Landsbury A, Dahm R, Liu YZ, Zhang QJ, Quinlan RA: Functions of the intermediate filament cytoskeleton in the eye lens. J Clin Invest 2009, 119:1837-1848. 58. Mencarelli C, Ciolfi S, Caroti D, Lupetti P, Dallai R: Isomin: a novel cytoplasmic intermediate filament protein from an arthropod species. BMC Biol 2011, 9:17. 59. Crick FHC: The packing of alpha-helices: simple coiled-coils. Acta Crystallogr 1953, 6:689-697. 60. Gruber M, Lupas AN: Historical review: another 50th anniversary – new periodicities in coiled coils. Trends Biochem Sci 2003, 28:679-685. 61. Kuhnel K, Jarchau T, Wolf E, Schlichting I, Walter U, Wittinghofer A, Strelkov SV: The VASP tetramerization domain is a righthanded coiled coil based on a 15-residue repeat. Proc Natl Acad Sci U S A 2004, 101:17027-17032. 62. Brown JH, Cohen C, Parry DA: Heptad breaks in alphahelical coiled coils: stutters and stammers. Proteins 1996, 26:134-145.

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