Abstract
Intermediate filament (IF) proteins have been predicted to have a conserved tripartite domain structure consisting of a largely alpha-helical central rod domain, flanked by head and tail domains. However, crystal structures have not been reported for any IF or IF protein. Although progress has been made in determining central rod domain structure, no structural data have been reported for either the head or tail domains. We used site-directed spin labeling and electron paramagnetic resonance to analyze 45 different spin labeled mutants spanning the head domain of vimentin. The data, combined with results from a previous study, provide strong evidence that the polypeptide backbones of the head domains form a symmetric dimer of closely apposed backbones that fold back onto the rod domain, imparting an asymmetry to the dimer. By following the behavior of spin labels during the process of in vitro assembly, we show that head domain structure is dynamic, changing as a result of filament assembly. Finally, because the vimentin head domain is the major site of the phosphorylation that induces disassembly at mitosis, we studied the effects of phosphorylation on head domain structure and demonstrate that phosphorylation drives specific head domain regions apart. These data provide the first evidence-based model of IF head domain structure.
Highlights
Mutations in Intermediate filament (IF) genes have been implicated in more than 85 different human diseases, with most diseases resulting from similar changes to highly conserved amino acids [6]
IF proteins can be solubilized in chaotropes such as 8 M urea, but upon dialysis into physiologic conditions, they spontaneously assemble into IFs
They are united into a family, on the basis of several features. Among these features are: (a) cytoplasmic IF proteins are typically found in 8 –11 nm IFs, and (b) in silico analysis of primary sequences predicts that all IF proteins possess a tripartite domain structure consisting of a central rod domain flanked by head and tail domains
Summary
A given spin mutant yields only very local structure within the complex, a series of mutants can be used to assemble a much more comprehensive view of a given protein or macromolecular complex. Using this approach, we have been able to define ␣-helical coiled-coil domains, establish spatial relationships between monomers in a dimer, and establish relationships between dimers in tetramers ( points of overlap and relative parallel/anti-parallel orientation), as well as the impact of disease-causing mutations and phosphorylation on IF structure [41,42,43,44,45,46]. We show some of the effects of both assembly and phosphorylation on IF head domain structure
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