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 regar ding 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 amin o acids are distributed periodically such that positively and negative ly charged residues are arranged in alternating zones, 9.5/2 residues wide (Parry et al., 1977; McLachlan and Stewart, 1982). This structura l feature has given rise to the hypothesis that, if neighboring coiled coils were staggered axially by an odd multiple of a charged zone, el ectrostatic 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 ass embly properties by DNA transfection into IF null cells, in vitro asse mbly and chemical cross-linking. Results from these experiments are co nsistent with, and support, the hypothesis that charge complementation plays a key role in the assembly and stabilization of intermediate fi laments.