All intermediate filament proteins possess three distinct domains: heads, rod and tail, and subdomains within the rod called helices 1A, 1B, 2A, and 2B. Subunit packing within a filament is a consequence of interactions among these domains. Several such interactions are known, but probably many more contribute to stabilizing filament structure. We examined a number of such potential interactions using the yeast two-hybrid system. Domains or subdomains of murine vimentin, a Type III intermediate filament protein, were fused with either the DNA-binding or trans-activating domain of GAL4, a transcription factor. Interaction between the vimentin domains/subdomains functionally reconstituted GAL4, thereby activating transcription of a GAL1-LacZ reporter gene. The oligomeric state at which the interactions took place, i.e. whether the domains/subdomains were dimeric or tetrameric as they interacted, was also determined. These studies revealed a number of interesting interactions, among which was a strong homotypic binding of helix 2B to form tetramers. They also demonstrated a lack of interaction among others expected to do so based on current structural models. From these results we deduced which of the candidates for interactions, suggested by current models, were true protein-protein interactions and which represented nearest-neighbors only. Thus, the A and A modes of molecular alignment identified by Steinert et al. (Steinert, P. M., Marekov, L. N., Fraser, R. D. B., and Parry, D. A. D.(1993) J. Mol. Biol. 230, 436-452) are probably true interactions, whereas the A and A modes may describe adjacent but non-interacting molecules.

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