Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly

Abstract

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

Fig. 1.

Primary structure analysis of the IF rod domain. (A) Sequence alignment for the rod domains of human type III IF proteins including vimentin, desmin, GFAP, and peri-pherin. Residues that are predicted by the NetSurfP algorithm (23) to be buried inside the structure are highlighted in yellow. Heptad assignment is indicated. The secondary structure prediction for the vimentin sequence by the Jpred3 algorithm (22) is shown on the Top (H indicates α-helix, E indicates β-sheet). Stutter inserts resulting in 11-residue repeats are highlighted with cyan. Proline residues are highlighted with gray. (B) Schematic diagram of coil 1 of human vimentin and its fragments discussed in the text. Yellow rectangles indicate the α-helical regions. The disordered region in the 1AB fragment structure is indicated by a dashed line. Red star indicates the Y117L mutation.

Fig. 2.

Crystal structures of the 1AB and 1ABL fragments. (A) Ribbon diagram of the 1AB structure. The side chains of residues in heptad positions a and d are shown in magenta. With the exception of residues 144 to 149 (shown in orange) in chain A, the rest of the linker L1 and coil 1A is disordered in the crystals. Figure drawn using the program Pymol (40). (B) Ribbon diagram of the 1ABL structure showing two chains A and B present in the crystallographic asymmetric unit. The 1A, L1, and 1B parts are colored yellow, orange and green, respectively. Within coil 1A, the residues in predicted heptad positions a and d (Fig. 1A) are shown in cyan and red, respectively (same as in E below). Within coil 1B, the residues in both heptad positions a and d are shown in magenta. The mutated Leu117 residue is labelled. (C) Ribbon diagram of the 1ABL structure showing chains A and B as well as a crystal symmetry-related copy (B’) of the latter. Chains B and B’ make contacts with chain A within coil 1B and coil 1A parts, respectively. The residues that are involved in the interaction of the coil-1A parts of chains A and B’ are shown in cyan; these correspond to heptad positions a and e in chain A and g and d in chain B’ (see also E). Correspondingly, there is a register shift of one residue between the parallel chains A and B’. (D) An antiparallel CC tetramer formed by the 1A parts in the 1ABL crystals. Chain A’ is a crystal symmetry equivalent of chain A. Chains B’ and B’’ are symmetry equivalents of chain B. For clarity, other symmetry-related chains forming the CC within the 1B part are not shown here. The residues involved in the hydrophobic core of the tetramer are shown in red, cyan, and gray according to the pairing indicated in E. In particular, the “knobs-to-knobs” interface of the antiparallel chains A and A’ is formed by five Leu residues on each side (all in positions d) and is shown with their side chains in red. The interface of antiparallel chains B’ and B’ is of a classical (antiparallel) ‘knobs-into-holes’ type; it involves the residues in a and e positions of either chain. (E) Alignment of the four 1A parts forming the antiparallel tetramer shown in D. The heptad assignment is indicated below each chain. The opposing pairs of residues forming the hydrophobic interactions (“knobs-into-holes”) between chains A and B’ and chains A’ and B’’ (parallel, one residue register shift) are shown in cyan. Similar interactions formed by chains B and B’ (antiparallel) are highlighted with gray. The knob-to-knob interface of antiparallel chains A and A’ is highlighted with red.

Fig. 3.

Complete structure of vimentin coil 1 based on crystal structures. (A) Least-squares structural superposition of the 1AB, 1ABL, and 1B fragments as well as the PDB entry 3UF1. The four structures are shown in blue, yellow, red and green respectively. (B) Plots of the CC radius (solid lines) and pitch (dash) as a function of residue number calculated using the program Twister (41). The data for the 1ABL, 1AB, 1B, and 3UF1 structures are shown in the same colors as in A.

Fig. 4.

Vimentin tetramer structure and lateral association mechanism. (A) Three-dimensional structure of vimentin dimer. The coil-1 structure (with coil 1A, L1, and coil 1B shown in yellow, orange, and green, respectively) is based on the superposition of the crystal structures shown in Fig. 3A. Similarly, the coil-2 structure (blue) is a superposition of previously established fragment structures (19, 42). The linker L12 for which no crystallographic data are available yet is shown schematically in pink. In the full dimer, the rod domain is flanked by the flexible head and tail domains, also without crystallographic information. (B) Three-dimensional structure of vimentin tetramer. Two antiparallel dimers were aligned in the A₁₁ mode following the tetrameric arrangement seen in the crystal structure (PDB entry 3UF1). (C) Molecular surfaces of two vimentin dimers forming the tetramer, colored by electrostatic potential (as estimated using the program Pymol). Blue and red coloring corresponds to positive and negative potential, respectively. Compared to B, one dimer (CD) was rotated by approximately 20° about the horizontal axis to reveal the surface facing the other dimer. The surface representation of this other dimer (AB) was additionally rotated by 180° to reveal the surface facing the dimer CD. The rectangle indicates the interaction areas presented in D. (D) Zoom-in to the interactions between the linker L1 region of chains A and B (shown as ribbons) and the C-terminus of coil 1B of dimer CD (shown as surface colored by electrostatic potential). All charged side groups of chains A and B are shown as sticks. (E) A possible involvement of parallel coil-1A segments in higher lateral assembly (octamers and beyond). If two tetramers ABCD and A’B’C’D’ (B) are laterally aligned, the dimers that run parallel to each other (AB and A’B’) may interact via a “cross-coil” formation of the coil-1A segments. This possibility is demonstrated by fitting the coil-1A parts of chains A and B’ on either chain of the dimeric coil-1A (Y117L) structure (PDB entry 3G1E, cyan).