The intermediate filament protein consensus motif of helix 2B: Its atomic structure and contribution to assembly

Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain. We have analyzed its contribution to the various stages of ass embly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them w ith the wild-type and tailless proteins. It is surprising that in buffers o f low ionic strength and high pH where the full-length proteins form tetram ers, both VimIAT and DesIAT associated into various high molecular weight c omplexes. After initiation of assembly, both VimIAT and DesIAT aggregated i nto unit-length-type filaments, which rapidly longitudinally annealed to yi eld filaments of around 20 nm in diameter. Mass measurements by scanning tr ansmission electron microscopy revealed that both VimIAT and DesIAT filamen ts contained considerably more subunits per cross-section than standard int ermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control o f filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amin o-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradu ally separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis. (C) 2000 Acad emic Press.