Charge interactions in the rod domain drive formation of tetramers during intermediate filament assembly.
The purpose of this study was to test a long standing hypothesis regarding the forces that drive the assembly of intermediate filaments (IFs). The initial step of IF assembly is the formation of dimeric, alpha-helical coiled coils. On the outside of the coiled coils, charged amino acids are distributed periodically such that positively and negatively charged residues are arranged in alternating zones, 9.5/2 residues wide (Parry et al., 1977; McLachlan and Stewart, 1982). This structural feature has given rise to the hypothesis that, if neighboring coiled coils were staggered axially by an odd multiple of a charged zone, electrostatic interactions between them could provide the driving force for the assembly of higher order oligomers or filaments (Fraser et al., 1986; Parry and Steinert, 1992). Using the IF protein vimentin as a model system, we carried out deletion mutagenesis experiments to test this hypothesis. We generated mutant vimentin proteins lacking 14, 21, and 28 residues in Helix 1B of the rod domain, and analyzed their assembly properties by DNA transfection into IF null cells, in vitro assembly, and chemical cross-linking. Results from these experiments are consistent with, and support, the hypothesis that charge complementation plays a key role in the assembly and stabilization of intermediate filaments.
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, Ohio 45267-0521.
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli; D005354:Fibrosarcoma; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000648:ultrastructure; D046911:Macromolecular Substances; D051379:Mice; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000648:ultrastructure; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000648:ultrastructure*
D018076:DNA, Complementary; D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014746:Vimentin