Featured Article - October 2012
Short description: Structural analysis of an intermediate filament protein reveals how an asymmetric dimeric coiled-coil assembles into a symmetric tetrameric structure.
The mammalian cytoskeleton is composed of filaments assembled from three different protein families. While microfilaments and microtubules are assembled from globular actin and tubulin protomers, respectively, intermediate filaments (IFs) follow a different structural paradigm and are assembled into coiled-coil (CC) oligomers from elongated protomer structures. As a result, IF proteins have biophysical properties that make structure determination challenging; to date no structure of an intact IF is available. Numerous IF proteins are expressed in tissue and development-specific patterns, highlighting the need for structural characterization of this diverse protein family.
FitzGerald, Hunt and colleagues (PSI NESG) have investigated the assembly and structure of two human IF protein vimentin domains using electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography. Sequence analysis of the central region of vimentin indicated four rod domains (1A, 1B, 2A and 2B) containing a heptad repeat pattern characteristic of a-helical coiled-coils. Small non-CC linkers interrupt the repeat pattern between the rod domains, although the exact boundaries and flexibility of these linkers was not obvious from the primary sequence.
The authors used EPR and exhaustive site-directed spin-labeling (SDSL) to investigate the dynamical properties of the vimentin linker 1 (L1) connecting rod domains 1A and 1B. They found that while L1 does indeed deviate from the CC structure it is not disordered and maintains a relatively rigid connection between the rod domains. Electron microscopy analysis of filament assembly by L1 mutants supports the conclusions from the EPR experiments.
To address the assembly of vimentin at atomic resolution, the authors solved the crystal structure of an L1-1B construct to 2.8-Å resolution (PDB 3UF1). The structure displays a homotetrameric assembly in which the subunit interactions are consistent with earlier EPR experiments. The tetramer is composed of two asymmetric parallel CC dimers with minor deviations from canonical CC geometry. Two conformationally inequivalent protomers form an asymmetrical parallel coiled-coil homodimer, and two of these associate in an antiparallel geometry to form a homotetramer with proper two-fold symmetry (i.e., perpendicular to the coiled-coil axis). The authors note that the asymmetry of the constituent dimers is essential for formation of the tetramer and for preventing further oligomerization. They further suggest that the vimentin sequence has evolved to exploit protein plasticity to favor the tetramer as the physiologically relevant assembly. As IF proteins are among the most abundant in eukaryotic cells and are associated with numerous diseases, future structural studies on vimentin and other IFs will explain the effects of pathogenic mutations.
A. Aziz et al. The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.
J. Biol. Chem. 287, 28349-28361 (2012). doi:10.1074/jbc.m111.334011