Structural Dynamics of the Vimentin Coiled-coil Contact Regions Involved in Filament Assembly as Revealed by Hydrogen-Deuterium Exchange

Abstract

Edited by Norma AllewellIntermediate 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 ␣-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 ␣-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 ␣-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 "crosscoiling" as the first step of the assembly process. Intermediate filament (IF)3 proteins form highly resilient filaments that are markedly resistant to mechanical stress. Mediated by prominent cytolinker proteins of the plakin family and motor proteins, they integrate actin filaments and microtubules to establish a functional cytoskeleton in metazoan cells and promote optimal tissue function (1). Apart from their basic mechanical function in maintaining cell flexibility, they are also involved in multiple cellular activities that range from cell division and motility to the topological organization of transmembrane channels (2, 3). Because of these multifunctional properties of IF proteins, mutations in IF-encoding genes cause almost 100 different inherited diseases in humans (4, 5). One of the most studied IF proteins, vimentin, forms IFs typical for mesenchymal cells, including endothelial cells, lymphocytes, and the eye lens epithelium (6). Of note, vimentin has long been implicated in many aspects of cancer initiation and progression, including tumorigenesis, epithelial-mesenchymal transition, and the metastatic spread of cancer (7), making this protein an attractive potential target for cancer diagnosis and therapy (8). Therefore, to develop mechanistic insight into the beh...

Quantitative SUMO proteomics identifies PIAS1 substrates involved in cell migration and motility

“…Under physiological conditions, VIM is maintained in equilibrium between ULF and soluble tetramers. The formation of VIM ULF has been shown to occur spontaneously on the order of seconds in vitro showing that this arrangement is thermodynamically favorable and proceeds rapidly without the need for protein modifications 50 . VIM filaments elongate by end-to-end annealing of ULFs and eventually form mature VIFs.…”

Lateral Subunit Coupling Determines Intermediate Filament Mechanics

“…Concerning aspect (iii), in contrast to keratin, vimentin IFs compact after elonga- tion [43,44]. Data from hydrogen-deuterium exchange show that for this compaction, charged amino acids in linker L1 and in coil 1B attract oppositely charged amino acids in linker L12 from a neighboring tetramer, which causes an additional link between these two tetramers [44]. Therefore, compaction can additionally increase the lateral coupling strength in vimentin IFs.…”

Human keratin 1/10‐1B tetramer structures reveal a knob‐pocket mechanism in intermediate filament assembly

“…It is evident from our model, as well as in cross‐linking studies (Steinert et al , ,b,c), that coils 1A and 2A also play a role in A 11 tetramer stabilization and intermediate filament formation. This was confirmed by hydrogen–deuterium exchange experiments on vimentin filament assembly; the stability of coils 1A and 2A increased during filament formation (Premchandar et al , ). Importantly, hydrogen–deuterium exchange also identified the N‐ and C‐termini of vimentin coil 1B as the most stable segments of the entire vimentin tetramer.…”

“…The mechanism of A 11 axial alignment in the keratin 1B region utilizes precise molecular interactions, which raises questions as to how KIFs utilize other predicted modes of alignment, such as the A 22 , A CN , or A 12 modes. We propose a simplified model where the A 11 tetramer is the building block for KIF assembly based on its strength, stability, and molecular precision as observed in our data and as observed in previous studies (Aziz et al, 2012;Herrmann & Aebi, 2016;Kim et al, 2018;Mü cke et al, 2004;Premchandar et al, 2016;Steinert et al, 1993a;Steinert et al, 1993b;Fig 9). Both the A 22 and A CN alignments essentially describe how an A 11 tetramer packs onto itself during the longitudinal elongation of tetramers into a protofilament (i.e., establishing length).…”