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Abstract

Previous studies have shown that the non−alpha−helical, amino−terminal head region of vimentin is essential for the formation and stability of vimentin intermediate filaments (IFs). In order to specify its target site on companion protein subunits, it was cut off from vimentin at amino acid position 96 with lysine−specific endoproteinase and allowed to react with intact vimentin and other IF proteins. In solution of high salt concentration (500 mM KCl), the isolated polypeptide (vim NT) showed a high affinity for all cytoplasmic IF proteins tested, but not for nuclear lamins. Employing limited digestion of the IF proteins with different proteinases, the binding site was shown to reside in their alpha−helical rod domains. Other polypeptides possessing alpha−helical regions with the potential to form coiled−coil structures like tropomyosin and myosin subfragment 2 did not react with vim NT. The binding to IF proteins was strongly inhibited by phosphorylation of vim NT and totally abolished in the presence of 200 mM arginine hydrochloride, whereas the same concentration of lysine hydrochloride was ineffective. Limited chymotryptic digestion of vim NT produced polypeptides that were unable to react with the alpha−helical region of vimentin at high salt concentration. Consistent with these observations, vim NT strongly inhibited filament formation in vitro from protofilamentous vimentin. A 14−mer oligopeptide comprising the amino acids 3 to 16 of the amino terminus also inhibited filament formation, though to a lesser extent. Conversely, vim NT and, with a lower efficiency, the 14−mer oligopeptide also severely affected the structure of preformed vimentin filaments by unraveling them. Phosphorylated vim NT was considerably less active in this respect. Further digestion of the rod domain of vimentin with chymotrypsin yielded 17.4 and 21 kDa polypeptides, which were tentatively characterized as originating from the carboxy− and amino−terminal half of the rod domain, respectively. Both formed salt−stable complexes with vim NT, the smaller polypeptide with a higher efficiency than the larger one. These results suggest that the staggered, antiparallel arrangement of the two coiled−coils in the protofilaments of IF proteins is, at least in part, determined by the twofold, symmetrical association of the amino−terminal head regions of one coiled−coil rope structure with the carboxy−terminal halves of the alpha−helical rod domains of the other coiled−coil and that similar interactions occur during filament assembly and in the intact filament