Program
| MSB3a | |
|---|---|
| Markus Meier | |
| Department of Chemistry, University of Manitoba | |
| Title | Biophysical characterization of vimentin coil 1A, a molecular switch |
| Abstract | Vimentin is an intermediate filament protein mainly found in fibroblast cells of the connective tissue. It has a tripartite structure consisting of a head domain, an α-helical rod domain and a tail domain. The rod itself is sub-segmented into four segments called coil 1A, coil 1B, coil 2A and coil 2B. The amino acid sequence contains a heptad repeat pattern that is characteristic for a coiled-coil structure. Interruptions ("stutters") in this pattern cause the sub-segmentation. The atomic structures of coils 1B, 2A and 2B are indeed dimeric parallel coiled coils (shown by the X-ray crystallography and/or analytical ultracentrifugation). Interestingly, the first published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric α-helical coil instead of the expected dimeric coiled coil1. However, the 39 amino acids long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutant variants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim to investigate the molecular criteria that are needed to stabilize the dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants did exhibit an increased stability over the wild type as indicated by a rise in the melting temperature (Tm). At a concentration of 0.1 mg/ml, the Tm of the peptide with the single point mutation Y117L increased dramatically by 46 ℃ compared to the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or d position did induce the formation of a stable dimer as we demonstrated by sedimentation equilibrium experiments. We also confirmed the dimeric oligomerisation state of the Y117L mutant peptide by X-ray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when we introduced this mutation into the full-length vimentin, the filament assembly was completely arrested at the unit-length filament (ULF) level, thus impeding the longitudinal elongation reaction. We concluded that the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might "switch" from a dimeric α-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the IF elongation reaction. References: 1. Strelkov, S. V., Herrmann, H., Geisler, N., Wedig, T., Zimbelmann, R., Aebi, U. & Burkhard, P. (2002). EMBO J. 21, 1255-1266. (in collaboration with G. Pauline Padilla, Harald Herrmann, Tatjana Wedig, Trushar R Patel, Jörg Stetefeld, Ueli Aebi, and Peter Burkhard) |
| Location | Woodward 3 |