Characterization of disulfide crosslink formation of human vimentin at the dimer, tetramer, and intermediate filament levels.
We have investigated the structural interactions of individual molecules of human vimentin in the soluble state and in filaments. Oxidative crosslinking experiments were conducted with wild-type vimentin aimed at the single cysteine in the helical domain coil 2b, mutated cysteine-free vimentin, and derivatives engineered to carry cysteines in presumed d positions of the heptad amino acid repeats in coils 1a and 2b. We provide conclusive evidence that crosslinking of the cysteine 328 in wild-type vimentin, when in the filamentous or tetrameric forms, occurs outside of the coiled-coil dimer, i.e., between staggered dimer molecules. This occurs despite the close axial register of the dimers and contradicts previous deductions. The extent of crosslinking increases with temperature as well as with the concentration of the crosslinking reagent. We conclude therefore that the cysteines are not in an ideal position for crosslinking but that molecular motion is needed to enhance the reaction. The occurrence of collision complexes, which has been speculated in the literature, does not occur and cannot explain these results. Furthermore, using tailless vimentin with the corresponding mutations, we provide compelling evidence that in type III intermediate filament proteins exchange of individual chains between dimers occurs only if the proteins are incubated in urea at concentrations above 3 M. In 5 M urea, however, the exchange is completed within seconds. The same reaction occurs between human vimentin and mouse desmin at a comparable speed, indicating that both type III intermediate filament proteins have a high affinity for one another at the coiled-coil level.
Rogers
K R
KR
Division of Cell Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Herrmann
H
H
Franke
W W
WW
D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents; D003893:Desmin / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D012995:Solubility; D014746:Vimentin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
D003432:Cross-Linking Reagents; D003893:Desmin; D004220:Disulfides; D014746:Vimentin