title: Head and rod 1 interactions in vimentin: identification of contact sites, structure, and changes with phosphorylation using site-directed spin labeling and electron paramagnetic resonance.
abstract: We have used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to identify residues 17 and 137 as sites of interaction between the head domain and rod domain 1A of the intermediate filament protein vimentin. This interaction was maximal when compared with the spin labels placed at up- and downstream positions in both head and rod regions, indicating that residues 17 and 137 were the closest point of interaction in this region. SDSL EPR characterization of residues 120-145, which includes the site of head contact with rod 1A, reveals that this region exhibits the heptad repeat pattern indicative of alpha-helical coiled-coil structure, but that this heptad repeat pattern begins to decay near residue 139, suggesting a transition out of coiled-coil structure. By monitoring the spectra of spin labels placed at the 17 and 137 residues during in vitro assembly, we show that 17-137 interaction occurs early in the assembly process. We also explored the effect of phosphorylation on the 17-137 interaction and found that phosphorylation-induced changes affected the head-head interaction (17-17) in the dimer, without significantly influencing the rod-rod (137-137) and head-rod (17-137) interactions in the dimer. These data provide the first direct evidence for, and location of, head-rod interactions in assembled intermediate filaments, as well as direct evidence of coiled-coil structure in rod 1A. Finally, the data identify changes in the structure in this region following in vitro phosphorylation.
authors.affiliation: Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616, USA.
authors.forename: Atya
authors.initials: A
authors.lastname: Aziz
authors.forename: John F
authors.initials: JF
authors.lastname: Hess
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Budamagunta
authors.forename: Paul G
authors.initials: PG
authors.lastname: FitzGerald
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
mesh_terms: D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D013113:Spin Labels; D014746:Vimentin / Q000737:chemistry*
chemical_list: D013113:Spin Labels; D014746:Vimentin