Title: Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly
Abstract: Article15 March 2002free access Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly Sergei V. Strelkov Corresponding Author Sergei V. Strelkov Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Harald Herrmann Harald Herrmann Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Norbert Geisler Norbert Geisler Division of Biochemistry and Cell Biology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, D-37070 Göttingen, Germany Search for more papers by this author Tatjana Wedig Tatjana Wedig Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ralf Zimbelmann Ralf Zimbelmann Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ueli Aebi Ueli Aebi Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Peter Burkhard Peter Burkhard Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Sergei V. Strelkov Corresponding Author Sergei V. Strelkov Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Harald Herrmann Harald Herrmann Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Norbert Geisler Norbert Geisler Division of Biochemistry and Cell Biology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, D-37070 Göttingen, Germany Search for more papers by this author Tatjana Wedig Tatjana Wedig Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ralf Zimbelmann Ralf Zimbelmann Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Ueli Aebi Ueli Aebi Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Peter Burkhard Peter Burkhard Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Author Information Sergei V. Strelkov 1, Harald Herrmann2, Norbert Geisler3, Tatjana Wedig2, Ralf Zimbelmann2, Ueli Aebi1 and Peter Burkhard1 1Maurice E.Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland 2Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 3Division of Biochemistry and Cell Biology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, D-37070 Göttingen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1255-1266https://doi.org/10.1093/emboj/21.6.1255 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF 'building block' is an elongated coiled-coil dimer consisting of four consecutive α-helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4–2.3 Å resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic α-helix, which is compatible with a coiled-coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer–dimer interactions during IF assembly. The 2B segment reveals a double-stranded coiled coil, which unwinds near residue Phe351 to accommodate a 'stutter'. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level. Introduction The cytoskeleton of higher eukaryotic cells contains three distinct types of filaments: microtubules, intermediate filaments (IFs) and microfilaments (Schliwa, 1986; Aebi et al., 1988; Fuchs and Weber, 1994; Herrmann and Aebi, 2000). The integrated network formed by these three filament systems together with various associated proteins is responsible for the mechanical integrity of the cell and is critically involved in such processes as cell division, motility and plasticity. While capable of self-assembly in vitro, the naturally occurring 10 nm wide IFs are dynamic structures that interact with other cytoskeletal components, in particular with motor proteins and plakin-type cross-bridging proteins (Herrmann and Aebi, 2000; Fuchs and Karakesisoglou, 2001). The crystal structures of tubulin and actin, the molecular 'building blocks' of microtubules and microfilaments, respectively, have been determined, and atomic models of the respective filaments have been built (Steinmetz et al., 1998; Gigant et al., 2000). However, until now, no atomic resolution structures of any IF component have been determined. The multigene family of IF proteins includes >60 members, which are grouped into four major sequence homology classes representing cytoplasmic IF proteins and a separate class representing the nuclear lamins (Conway and Parry, 1988; Herrmann and Aebi, 1998a). The amino acid sequences of all IF proteins share a characteristic tripartite structure (Figure 1A) that includes the central highly α-helical 'rod' domain flanked by the 'head' and 'tail' domains at both ends (Geisler and Weber, 1982; Fuchs and Weber, 1994). The rod domain reveals a heptad repeat pattern, which is a signature of a coiled-coil (CC) fold (see, for example, Lupas, 1996). Correspondingly, the elementary 'building block' underlying the IF architecture is an elongated, parallel CC dimer. The heptad periodicity within the rod domain is, however, interrupted in several places, resulting in four consecutive α-helical segments 1A, 1B, 2A and 2B that are connected by short linkers L1, L12 and L2. In particular, the rod domain of all vertebrate cytoplasmic IF proteins contains close to 310 residues, and the sizes of the individual α-helical segments are absolutely conserved (Fuchs and Weber, 1994; Parry and Steinert, 1999). In addition, both nuclear lamins and invertebrate IF proteins contain an insertion of six extra heptads in the segment 1B. Figure 1.(A) Primary structure of IF proteins. Schematic diagram of human vimentin. Rectangles show α-helical segments, including the pre-coil domain (PCD). (B) Sequence alignment of the 1A segments of human IF proteins including vimentin, desmin, neurofilament L protein, cytokeratins 8 and 18, and nuclear lamins A and B1. (C) Similar alignment of the 2B segments. Vimentin fragments 1A, Cys2, Z2B and 2B2 are highlighted. The heptad repeats are marked as abcdefg, with core positions highlighted with yellow. Basic and acidic residues are shown in blue and red, respectively. The line below the alignment shows the sequence similarity score s of a particular residue in the seven proteins: '*', s = 1.0 (absolutely conserved); 'x', 0.75≤s<1.0; ':', 0.5≤s<0.75; '.', 0.25≤s<0.5 (see Materials and methods for details). The two most conserved regions within the 1A segment and in the C-terminal part of the 2B segment, respectively, are shown in boxes. Download figure Download PowerPoint IF assembly begins with a gradual association of the dimers, governed by several distinct modes of lateral interaction (Steinert et al., 1993; Herrmann and Aebi, 1998a). This process first leads to the formation of tetramers (Herrmann and Aebi, 1999) and ultimately yields so-called unit-length filaments (ULFs). In particular, the vimentin ULFs appear to contain about sixteen ∼46 nm long dimers (Herrmann et al., 1996). Subsequently, the ULFs anneal longitudinally into rather loosely packed filaments. Finally, the extended filaments undergo an internal rearrangement of subunits which manifests itself by a radial compaction of the filament (Herrmann and Aebi, 1998b, 1999). Notably, the mature filament structure includes a short head–tail overlap between consecutive dimers. The resulting IFs appear to have no polarity, as the individual dimers are oriented along its axis in either direction. Finally, the head and tail domains play a critical role in the filament assembly process, despite the fact that they vary considerably in sequence and length among different IF proteins (Heins and Aebi, 1994; Herrmann et al., 1996). The sequence conservation across different IF proteins is especially pronounced within two regions that are located at the opposite ends of the CC rod (Figure 1B and C). The first conserved region spans 26 residues corresponding to about two-thirds of the α-helical segment 1A, and involves eight residues which are absolutely conserved within the sequence comparison presented in Figure 1. The second conserved region is situated at the very end of the 2B segment. This region spans 32 residues, of which 13 are absolutely conserved, and harbors the so-called IF 'consensus' motif YRKLLEGEE (Herrmann et al., 2000). Chemical cross-linking studies have revealed that both of these highly conserved regions are critically involved in various dimer–dimer interactions within the mature filament (Steinert et al., 1993; Wu et al., 2000). The significance of the two conserved regions is supported further by the fact that they harbor a number of phenotypically pronounced point mutations (Parry and Steinert, 1999). These include, in particular, hereditary defects in keratins which were linked to various skin blistering diseases in humans, such as epider molysis bullosa simplex (for reviews see Fuchs and Coulombe, 1992; Fuchs and Weber, 1994; Parry and Steinert, 1999). In addition, the 2B segment contains a discontinuity in the heptad repeat pattern, a so-called 'stutter', which is equivalent to an insertion of four extra residues at the end of a heptad (Brown et al., 1996; Lupas, 1996). The stutter appears to be an obligatory feature in all IF proteins (Weber and Geisler, 1985), and its position at the end of the eighth full heptad of 2B is absolutely conserved despite a high sequence variability in this region (Figure 1C). The major obstacle preventing high-resolution X-ray studies of IFs has always been the difficulty in growing suitable three-dimensional crystals. The breakthrough came after we suggested that, instead of crystallizing the full-length IF protein, one should use multiple overlapping fragments thereof. Following this 'divide-and-conquer' strategy, we recently have obtained X-ray quality crystals from six fragments of human vimentin (Herrmann et al., 2000; Strelkov et al., 2001). Here we present the atomic structures of three such fragments as well as accompanying biochemical experiments on IF assembly. Taken together, these data reveal the molecular architecture and function of the highly conserved α-helical segments 1A and 2B of the IF dimer. Results Crystallographic structure determination We have obtained crystal structures of three fragments of human vimentin, named 1A, Z2B and Cys2, respectively (Figure 1B and C). The design and crystallization of these fragments were described previously (Strelkov et al., 2001). The 1A fragment contains residues 102–138 corresponding to the first α-helical segment of the vimentin rod. Z2B is a chimeric peptide which includes vimentin residues 385–412, i.e. the highly conserved region of the 2B segment, fused at the N-terminus to the 31 residue leucine zipper domain from the yeast transcription activator GCN4 (O'Shea et al., 1991). This fusion was designed to provide for formation of a proper double-stranded CC within the relatively short vimentin sequence (Herrmann et al., 2000). Finally, the Cys2 fragment (residues 328–411) includes the major part of the α-helical segment 2B starting with residue Cys328. It therefore contains the vimentin part of the Z2B chimera within itself. X-ray diffraction data for all three vimentin fragments were collected at resolutions between 1.4 and 2.3 Å (Table I). The 1A and Z2B crystals contain a monomeric α-helix and a double-stranded CC, respectively, per asymmetric unit (Figures 2A and 3A). Both structures were phased by molecular replacement (MR) using models derived from the GCN4 leucine zipper. While MR is generally known to be difficult for 'rod-like' molecules (Turkenburg and Dodson, 1996), the case of the 1A fragment was complicated further by the fact that an α-helical structure repeats itself after a screw rotation about the helix axis. Since a CC possesses a similar property, the MR searches for the Z2B construct were also rather elaborate despite the presence of the exact GCN4 leucine zipper sequence within the molecule. The diffraction data for the Cys2 fragment could only be phased by multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives. The Cys2 crystals contain three independent, ∼120 Å long CC dimers per asymmetric unit, denoted AB, CD and EF, respectively. The dimers are aligned approximately along the longest unit cell axis c (Figure 3B). All three crystal structures were refined to good crystallographic R-factors while securing quality stereochemical parameters (Table I). Figure 2.Crystal structure of the vimentin fragment 1A. (A) Stereo view of the atomic model and electron density map with coefficients 2Fobs − Fcalc contoured at 1.2σ. Residues in the a and d positions of the putative heptad repeat are shown in magenta. Solvent molecules are shown as blue spheres. (B) The crystal packing arrangement of 1A shown in stereo. The a and d positions are highlighted with magenta. The N- and C-termini of the helices are marked in red and blue, respectively. (C) Modeling of a parallel coiled-coil by docking two 1A helices (red) while using the GCN4 zipper structure (cyan) as a ruler. Download figure Download PowerPoint Figure 3.Crystal structures of the Z2B and Cys2 fragments. (A) Ribbon diagram of the Z2B structure. The GCN4 leucine zipper and the authentic vimentin residues are shown in magenta and yellow, respectively. (B) Ribbon diagrams of the three symmetry-independent Cys2 dimers AB (red), CD (green) and EF (blue) in the unit cell. The position of the stutter in each dimer is marked with an asterisk. Download figure Download PowerPoint Table 1. Crystallographic data Fragment name 1A Z2B Cys2 Vimentin residues included 102–138 385–412 328–411 No. of residues per chaina 39 59 84 Diffraction data Space group P6222 P3121 I222 Cell constants a × b × c (Å) 56.8 × 56.8 × 58.4 98.8 × 98.8 × 36.5 76.6 × 84.3 × 240.8 Resolution limitsb,c, b,c (Å) 50.0–1.4 (1.45–1.40) 35.0–1.9 (1.97–1.90) 35.0–2.3 (2.33–2.30) No. of independent reflections 11 440 (1122) 15 422 (1604) 34 937 (1159) Redundancy 10.3 (5.5) 4.0 (3.7) 4.5 (4.0) Completeness (%) 99.7 (99.6) 99.2 (99.4) 99.5 (99.7) <I/σ> 17.0 (1.9) 14.6 (3.3) 13.5 (1.8) Rsymd 0.037 (0.560) 0.063 (0.372) 0.058 (0.405) Refined model Protein chains/asymmetric unit 1 2 6 Ordered vimentin residues in each chain A: 102–138 A: 385–409 A: 328–406 B: 385–406 B: 328–406 C: 337–406 D: 330–407 E: 337–406 F: 333–406 No. of solvent molecules 35 194 455 Total no. of non-H atoms 375 1066 4170 Average model B-factor 23.5 31.5 53.8 Rworke 0.197 0.199 0.242 Rfreee,f, e,f 0.216 (461) 0.227 (778) 0.262 (1095) R.m.s.d. bondsg (Å) 0.020 0.017 0.008 R.m.s.d. anglesg (°) 2.0 1.7 1.1 a Besides the authentic vimentin residues, the 1A fragment contains two extra residues, GlySer, at the N-terminus (see Strelkov et al., 2001). The Z2B chimera includes the GCN4 leucine zipper. b Data in parentheses are for the highest resolution shell. c Cys2 crystals exhibited anisotropic diffraction, which extended up to 1.9 Å resolution in the c* direction and to 2.3 Å resolution in the a* and b* directions. d , where Ihi is the ith intensity measurement of a reflection with index h. e R = Σ|Fobs − Fcalc|/∑Fobs. f In parentheses is the number of randomly selected reflections that were excluded from refinement and used to calculate the 'free' R-factor (Brünger and Nilges, 1993). g R.m.s.ds from the Engh and Huber standard parameters. Atomic structure of the 1A segment We have found previously that the recombinant vimentin fragment 1A is monomeric in solution, while its circular dichroism spectra reveal ∼30% α-helical and 70% random structure (Strelkov et al., 2001). Most strikingly, the 1A crystals also contain a single polypeptide chain per asymmetric unit (Figure 2A). Furthermore, all of its 39 residues are strictly α-helical (φ approximately −65°, ψ approximately −45°). As a consequence of the pronounced heptad repeat pattern, most apolar side chains locate approximately on one side of the relatively short α-helix. Interestingly, the helix is slightly bent (radius of curvature r = 84 Å) in such a way that the hydrophobic patch locates on the concave side of the curvature. Furthermore, the 1A crystal packing arrangement reveals a 'layered' pattern (Figure 2B). Each layer is formed by alternating antiparallel 1A helices located in a plane perpendicular to the crystallographic 6-fold axis. The residues in the a and d positions are situated on one side of the layer, rendering this side substantially more hydrophobic than the other. Moreover, the orientations of consecutive layers alternate between 'up' and 'down'. As a result, distinct 'bilayers' are observed, with a hydrophobic interface between the two layers within a bilayer. The crossing angle between the axes of helices in the two layers is 45°. Notably, about three-quarters of all ordered solvent molecules are located on the hydrophilic side of the α-helix (see Figure 2A) and thus between the adjacent bilayers. The crystal packing arrangement of the 1A fragment does not reveal CCs of any kind (parallel or antiparallel, aligned in register or staggered). However, the bending of the 1A helix results in a conformation which is fully compatible with a double-stranded parallel CC geometry. This can be shown by fitting two 1A helices as rigid bodies onto either chain of the GCN4 leucine zipper, with heptad repeats in phase (Figure 2C). After some adjustment of the side chain conformation in the a and d positions, such an artificial dimer would have a perfect CC structure. Furthermore, the crystal structure of the 1A fragment does not reveal any intrahelical salt bridges. However, within the constructed dimer, an interhelical salt bridge between Lys120 (heptad position g) and Glu125 (position e′ of the following heptad) is feasible. Last but not least, these data suggest that, generally speaking, the curvature of an α-helix within a CC is not a consequence of the hydrophobic core formation, as may be thought. Instead, it appears to be an intrinsic feature of an α-helix with most apolar side chains localized on one side. However, this hypothesis requires further experimental confirmation. Atomic structure of the 2B segment The crystal structures of the Z2B and Cys2 fragments document that the 2B segment of vimentin forms a continuous double-stranded CC (Figure 3A and B). Importantly, both structures independently indicate that this segment terminates with residue Glu405. While the latter is the last residue in an α-helical conformation, Leu404 in an a position is the last residue involved in the hydrophobic seam between the two helices. The residues past Gly406 appear to be disordered in most of the cases. An important exception is one chain of the Z2B construct which reveals that the residues Glu407, Glu408 and Ser409 fold back onto the CC away from its axis (Figure 3A, see also Figure 4B). At the same time, a least-squares superposition of the three crystallographically independent Cys2 dimers and the Z2B structure reveals some variability in the overall geometry of the CC (Figure 4A) and also considerable differences in the side chain conformation of certain residues (Figure 4B). This points to the inherent conformational flexibility of the CC structure. In addition, as documented in Table I, it turns out that only one of the three Cys2 dimers, AB, includes an ordered CC starting with the N-terminal Cys328 residues of both chains. The two other dimers, CD and EF, reveal a proper CC geometry starting only with residue Leu340 in a d position, while the preceding residues are partially disordered. In particular, in the electron density maps, both chains C and E are only traceable starting with residue Asn337, and their residues 337–339 are clearly folding away from the CC axis (Figures 3B and 4A). While the differences between the three dimers are due to a different crystallographic environment, these data suggest that the CC between residues 328 and 340 is relatively labile. Figure 4.Superposition of the Cys2 and Z2B structures. (A) Ribbon diagrams of the three Cys2 dimers (AB, red; CD, green; and EF, blue) and the Z2B dimer (yellow). (B) The C-terminal part of the 2B segment shown in stereo (coloring as above). Salt bridges are shown with dotted lines. Download figure Download PowerPoint Importantly, the Z2B and Cys2 structures reveal a network of intra- and interhelical salt bridges within the highly conserved C-terminal region of the 2B segment (Figure 4B). The first, intrahelical i to i + 4 type salt bridge, links residues Lys390 and Asp394 located in heptad positions a and e, respectively. This salt bridge is present in all eight symmetry-independent polypeptide chains of the two crystal structures. The second, g–e′ type interhelical salt bridge, is formed between the residues Glu396 and Arg401. This salt bridge was found only in three out of the eight cases. In the remaining five cases, the side chains of Glu396 and Arg401, while being visible in the electron density maps, are separated by distances that exclude effective ionic interactions (>4.0 Å). However, in two cases, the Arg401 residue forms an intrahelical i to i + 4 type salt bridge with Glu405 instead (Figure 4B). The Cys2 structure documents that the stutter occurring near the vimentin residue 351 can be tolerated without destroying the CC geometry (Figure 5A). Indeed, the two α-helices run continuously through the stutter site, and the main chain hydrogen bonding pattern is fully preserved as well. Interestingly, the two Phe351 rings are arranged asymmetrically with respect to the CC axis, and locate one after the other in the hydrophobic core. This arrangement appears to be possible due to the presence of the small Ala355 residue in the next core position (a). These phenylalanine and alanine residues are highly conserved in vimentins and desmins of vertebrates (Herrmann and Aebi, 1999), but not in more distant IF proteins (Figure 1C). Figure 5.Effect of the coiled-coil stutter within the 2B segment. (A) Hydrophobic core organization near the stutter of the Cys2 dimer. The letters in parentheses after the residue number indicate the heptad position. The dotted lines connect the Cα atoms of the consecutive core residues. (B) Coiled-coil radius (green) and pitch (blue) as a function of residue number. The data for the Cys2 structure (averages over three independent dimers) and Z2B structure are shown with solid and dashed lines, respectively. Download figure Download PowerPoint Geometrical parameters of a CC are often estimated globally by fitting an idealized CC structure to the experimental coordinates (O'Shea et al., 1991; Tao et al., 1997). We have developed a new algorithm to analyze the CC geometry in considerably more detail by directly determining each of these parameters locally as a function of residue number (see Materials and methods). Figure 5B shows the variability of the two principal parameters, namely the superhelix radius and pitch, within the 2B segment. Starting with residue 355, i.e. downstream of the stutter, the 2B segment reveals a fairly even CC geometry with a radius of 5.22 ± 0.24 Å and a pitch of 160.5 ± 43.0 Å (average values over the three Cys2 dimers). For comparison, Tao et al. (1997) have found an average radius of 5.0 Å and an average pitch of 128.5 Å for several typical double-stranded CCs. The stutter, however, results in a sharp local increase of the CC pitch (Figure 5B), which corresponds to the two α-helices becoming nearly parallel (cf. Figure 3B). In addition, there is a slight increase of the CC radius, while both the α-helical radius and pitch remain essentially constant (data not shown). It is therefore the local unwinding of the CC that chiefly compensates for the stutter. This observation is in accordance with earlier theoretical calculations (Brown et al., 1996). Averaged over the three Cys2 dimers, the unwinding corresponds to a 28.3° 'delay' of the CC phase, compared with a regular CC with a pitch of 161 Å for the region between residues 341 and 354 (cf. Figure 5B). Influence of the 2B2 fragment on IF assembly In addition to the X-ray crystallographic studies, we have examined the influence of a further vimentin construct, 2B2, incorporating residues 355–412, on IF assembly (Figure 1C). Like the Z2B and Cys2 fragments, this construct includes the highly conserved sequence at the C-terminal end of the rod and forms CC dimers in solution (Strelkov et al., 2001). We have evaluated, first, the co-assembly of wild-type human vimentin with the 2B2 fragment and, secondly, the effect of this fragment on the already assembled recombinant human vimentin IFs. In the absence of 2B2, addition of the 'filament buffer' to a solution of vimentin tetramers (see Materials and methods) resulted in the formation of bona fide IFs (Figure 6B). Upon centrifugation, the filaments went entirely into the pelleted fraction (Figure 6A, lanes 2 and 3). When the filament assembly was initiated in the presence of a 10-fold excess of 2B2, most of the wild-type vimentin was again found in the pellet, with only a trace amount remaining in the soluble fraction (Figure 6A, lanes 4 and 5). Remarkably, some of the 2B2 fragment also went into the pellet. Thus some kind of assembly does occur also in the presence of 2B2. However, electron microscopy of the pellets after the co-assembly already at a 1:1 molar ratio revealed flat, short and partially unraveled fibrillar structures (not shown), suggesting that IF elongation was substantially inhibited. Most strikingly, similar aberrant structures could be observed already within several minutes after the 2B2 fragment was added to pre-assembled vimentin IFs, as nearly all IFs were severed and transformed into short, ribbon-like fibers (Figure 6C). Taken together, these results indicate that the 2B2 fragment readily interferes with both vimentin tetramers and mature IFs. Figure 6.Effect of the 2B2 fragment on IF assembly. (A) Denaturing gel electrophoresis (SDS–PAGE) illustrating the assembly of human recombinant vimentin alone (lanes 2 and 3) and in the presence of the 2B2 fragment (lanes 4 and 5). The 2B2 fragment was added to the vimentin sample (lane 1) at a 10-fold molar excess, and then filament assembly in the test and reference (i.e. without the fragment addition) samples was performed as described in Materials and methods. The samples subsequently were centrifuged in a Beckman Airfuge for 30 min at 10 p.s.i. yielding the supernatant (lanes 2 and 4) and pelleted (lanes 3 and 5) fractions. The arrow indicates the location of the gel front. (B) Negatively stained EM images of vimentin IFs assembled in vitro. The samples were prepared by ultrathin sectioning (main figure) or on grids (inset). Scale bars are 100 nm. (C) Similarly assembled IFs, which subsequently were incubated with a 10-fold molar excess of the 2B2 fragment for 1 h at 37°C. Download figure Download PowerPoint Model of the IF dimer Taken together, the crystal structures of the 1A, Z2B and Cys2 fragments describe the molecular organization of the α-helical segments 1A and 2B. In addition, preliminary studies of another fragment corresponding to the 1B segment point to a double-stranded CC (A.Lustig and S.V.Strelkov, unpublished). Based on these data accompanied by homologous modeling, we have assembled a three-dimensional model of the vimentin dimer (Figure 7A and B). This model, in particular, illustrates the 'open' and 'closed' conformations of the 1A segments within the whole molecule (see Discussion for details). Inte