Vimentin coil 1A-A molecular switch involved in the initiation of filament elongation. Export

@article{citeulike:5660732, abstract = {Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a 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 exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T(m)). At a concentration of 0.1 mg/ml, the T(m) of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by X-ray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, 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 alpha-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 intermediate filament elongation reaction.}, author = {Meier, Markus and Padilla, G. Pauline and Herrmann, Harald and Wedig, Tatjana and Hergt, Michaela and Patel, Trushar R. and Stetefeld, J\"{o}rg and Aebi, Ueli and Burkhard, Peter}, citeulike-article-id = {5660732}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.jmb.2009.04.067}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/19422834}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=19422834}, day = {10}, doi = {10.1016/j.jmb.2009.04.067}, issn = {1089-8638}, journal = {Journal of molecular biology}, keywords = {coiled-coil, structure, vimentin}, month = {July}, number = {2}, pages = {245--261}, posted-at = {2009-08-28 00:36:11}, priority = {2}, title = {Vimentin coil 1A-A molecular switch involved in the initiation of filament elongation.}, url = {http://dx.doi.org/10.1016/j.jmb.2009.04.067}, volume = {390}, year = {2009} }

TY - JOUR ID - citeulike:5660732 L3 - citeulike-article-id:5660732 N2 - Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a 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 exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T(m)). At a concentration of 0.1 mg/ml, the T(m) of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by X-ray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, 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 alpha-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 intermediate filament elongation reaction. IS - 2 JF - Journal of molecular biology SN - 1089-8638 EP - 261 TI - Vimentin coil 1A-A molecular switch involved in the initiation of filament elongation. VL - 390 SP - 245 KW - coiled-coil KW - structure KW - vimentin AU - Meier, Markus AU - Padilla, Pauline AU - Herrmann, Harald AU - Wedig, Tatjana AU - Hergt, Michaela AU - Patel, Trushar AU - Stetefeld, Jörg AU - Aebi, Ueli AU - Burkhard, Peter PY - 2009/07/10/ UR - http://dx.doi.org/10.1016/j.jmb.2009.04.067 ER -