Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They are not only responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central α-helical domain ("rod"). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also， a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend th... More
Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They are not only responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central α-helical domain ("rod"). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also， a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and to investigate the dynamic properties of the full-length proteins in solution during the various assembly steps， we analyzed the patterns of hydrogen-deuterium exchange in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of the α-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within the full-length vimentin， in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers， ULFs， and mature filaments. Moreover， hydrogen-deuterium exchange analysis of the point-mutated variants directly demonstrated the active role of the IF consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately， using molecular dynamics simulation procedures， we provide a structural model for the subdomain-mediated tetramer/tetramer interaction via "cross-coiling" as the first step of the assembly process.
