Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

Abstract

<jats:p>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 ACN 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 ACN complex which features a 6.5 nm overlap of the rod ends.</jats:p>

Molecular Interactions Driving Intermediate Filament Assembly

“…Technical details, pearls, and possible pitfalls of crystallographic studies were discussed in [ 4 , 26 ]. One common challenge of shorter rod domain fragments is that they may not yield parallel, in-register dimeric coiled-coils [ 26 , 30 ]. Therefore, it is advisable to check for the correct fragment oligomerization by means of biophysical techniques such as gel filtration coupled to multi-angle light scattering.…”

“…The solution to this problem could be found in fusing the short fragments with either an N- or C-terminal capping motif that would ‘bootstrap’ the formation of a correct dimeric, parallel, and in-register coiled-coil. This approach had been originally developed for coiled-coil fragments of myosin [ 33 ] and more recently applied to nuclear lamins [ 30 ]. A particularly efficient capping motif may include a cysteine residue that forms a disulfide bridge across the two chains.…”

“…Toward crystallographic phasing, the capping motif alone is often sufficient to provide the initial phase estimate through molecular replacement. Alternatively, a disulfide bridge located on the dimer axis is capable of producing a relatively strong anomalous signal that can also be used for phasing [ 30 ].…”

“…In fact, these data have been widely regarded as establishing the basis of IF architecture ever since [ 8 , 56 ]. Interestingly, it is only in the last few years that new chemical cross-linking results became available, this time, on nuclear IFs [ 30 , 53 , 57 ]. In this section, we will cover both the technical aspects of the technique and its impact on understanding the IF structure.…”

Separation of Coiled-Coil Structures in Lamin A/C Is Required for the Elongation of the Filament

“…The parallel overlapping between the C-terminal region of coil 2 and the coil 1a region in the A11 tetramer was crucial for the eA22 interaction to form the eA22 binding mode. However, the overlapping length between the A11 tetramers in the eA22 binding mode is under debate [ 18 , 26 , 27 ]. Thus, further study is required to understand the molecular mechanism for the elongation of IF proteins.…”