title: Stabilization of vimentin coil2 fragment via an engineered disulfide.
abstract: Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficiently short. As a consequence, they often fail to assemble into the correct CC dimers. In particular, human vimentin fragment D3 corresponding to the first half of coil2 (residues 261-335) stays monomeric in solution. We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus. The 2.3 Å crystal structure of the D3st (stabilized) fragment reveals a mostly parallel α-helical bundle structure in its N-terminal half which smoothly continues into a left-handed CC towards the C-terminus. This provides a direct evidence for a continuously α-helical structure of the coil2 segment and disproves the previously suggested existence of linker L2 separating it into two left-handed CCs. The general principles of CC dimer stabilization by disulfide introduction are also discussed.
authors: Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit Leuven, Belgium.
authors: A A
authors: AA
authors: Chernyatina
authors: S V
authors: SV
authors: Strelkov
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014746:Vimentin / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure*
chemical_list: D004220:Disulfides; D014746:Vimentin