Laboratory for Biocrystallography, KU Leuven, 3000 Leuven, Belgium.
Department of Biochemistry, Charles University, 12800 Prague, Czech Republic.
Cells. 2020 Jul 7;9(7):1633. doi: 10.3390/cells9071633.
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 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 complex which features a 6.5 nm overlap of the rod ends.
中间丝的分子结构和组装机制几十年来一直是个谜。其中，核纤层蛋白因其在细胞核中的普遍作用和与许多疾病相关的突变而备受关注。纤维丝的组装是由基本二聚体的特异性相互作用驱动的，这些二聚体由中央的螺旋卷曲杆域和非螺旋的头和尾域组成。我们旨在研究核纤层蛋白二聚体的纵向“头对头”相互作用（即所谓的 A 相互作用），这对于纤维丝的组装至关重要。为此，我们制备了一系列以杆的 N 端和 C 端为中心的人核纤层蛋白 A 的重组片段。这些片段通过与异源帽状结构域融合而稳定，这为平行、对齐的螺旋卷曲二聚体的正确形成提供了条件。结果，我们建立了两个 N 端片段的晶体结构，其中一个突出了螺旋卷曲易于打开的倾向，还有一个 C 端杆片段。进一步的研究强调了这些 N 端和 C 端片段在溶液中形成特定复合物的能力，并用化学交联进一步对其进行了表征。这些数据得出了 A 复合物的分子模型，其特征是杆端重叠 6.5nm。
