Given the role of intermediate filaments (IFs) in normal cell physiology and scores of IF-linked diseases, the importance of understanding their molecular structure is beyond doubt. Research into the IF structure was initiated more than 30 years ago, and some important advances have been made. Using crystallography and other methods, the central coiled-coil domain of the elementary dimer and also the structural basis of the soluble tetramer formation have been studied to atomic precision. However, the molecular interactions driving later stages of the filament assembly are still not fully understood. For cytoplasmic IFs, much of the currently available insight is due to chemical cross-linking experiments that date back to the 1990s. This technique has since been radically improved, and several groups have utilized it recently to obtain data on lamin filament assembly. Here, we will summarize these findings and reflect on the remaining open questions and challenges of IF structure. We argue that, in addition to X-ray crystallography, chemical cross-linking and cryoelectron microscopy are the techniques that should enable major new advances in the field in the near future.

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal ‘head-to-tail’ interaction of lamin dimers (the so-called A_CN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the A_CN complex which features a 6.5 nm overlap of the rod ends.

All proteins of the intermediate filament (IF) family contain the signature central α-helical domain which forms a coiled-coil dimer. Because of its length, past structural studies relied on a ‘divide-and-conquer’ strategy whereby fragments of this domain were recombinantly produced, crystallized and analysed using X-rays. Here we describe a further development of this approach towards structural studies of nuclear IF protein lamin. To this end, we have fused lamin A fragments to short N- and C-terminal capping motifs which provide for the correct formation of parallel, in-register coiled-coil dimers. As the result, a chimeric construct containing lamin A residues 17-70 C-terminally capped by the Eb1 domain was solved to 1.83 Å resolution. Another chimera containing lamin A residues 327-403 N-terminally capped by the Gp7 domain was solved to 2.9 Å. In the latter case the capping motif was additionally modified to include a disulphide bridge at the dimer interface. We discuss multiple benefits of fusing coiled-coil dimers with such capping motifs, including a convenient crystallographic phasing by either molecular replacement or sulphur single-wavelength anomalous dispersion (S-SAD) measurements.