Structural Characterization of Nanoscale Meshworks within a Nucleoporin FG Hydrogel

Abstract

The permeability barrier of nuclear pore complexes (NPCs) controls all exchange of macromolecules between the cytoplasm and the cell nucleus. It consists of phenylalanine−glycine (FG) repeat domains apparently organized as an FG hydrogel. It has previously been demonstrated that an FG hydrogel derived from the yeast nucleoporin Nsp1p reproduces the selectivity of authentic NPCs. Here we combined time-resolved optical spectroscopy and X-ray scattering techniques to characterize such a gel. The data suggest a hierarchy of structures that form during gelation at the expense of unstructured elements. On the largest scale, protein-rich domains with a correlation length of ∼16.5 nm are evident. On a smaller length scale, aqueous channels with an average diameter of ∼3 nm have been found, which possibly represent the physical structures accounting for the passive sieving effect of nuclear pores. The protein-rich domains contain characteristic β-structures with typical inter-β-strand and interβ-sheet distances of 1.3 and 0.47 nm, respectively. During gelation, the formation of oligomeric associates is accompanied by the transfer of phenylalanines into a hydrophobic microenvironment, supporting the view that this process is driven by a hydrophobic collapse.

The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei

“…Gorlich and colleagues have reported similar observations as a function of maturation of liquid-like droplets formed from FG repeats associated with nucleoporin proteins (Petri et al ., 2012). In summary, mutational studies of the LC domain of hnRNPA2 give evidence that similar forces drive both hydrogel retention and partitioning into liquid-like droplets, and NAI footprinting studies reveal evidence that the LC domain of hnRNPA2 adopts a similar structure in both settings.…”

“…It is possible that the physical forces leading to the two states are entirely different. Recent studies of the DDX4 protein and LC domains associated with nucleoporin proteins characterized by FG repeats favor the utility of chemical interactions deployed to intertwine otherwise unstructured, random coil LC domains (Nott et al, 2015; Petri et al, 2012). In the case of DDX4, Pi stacking between arginine and phenylalanine residues has been highlighted as a key chemical determinant for phase separation into liquid-like droplets.…”

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

“…Amyloid‐like, intermolecular β‐sheets between NQ‐rich sequences are dominating structural elements in the hydrogel formed by the FG domain from S. cerevisiae Nsp1p (Ader et al , 2010; Petri et al , 2012). This coincides with a high NQ content (≈27%; Table I) of the most cohesive part of this domain (scNsp1 1–175 ) and of other highly cohesive yeast FG domains, such as those from scNup100p or scNup116p (Ader et al , 2010; Yamada et al , 2010; Halfmann et al , 2012).…”

Nuclear bodies: the emerging biophysics of nucleoplasmic phases

“…FUS contains an N-terminal LCS domain, which was found to drive a concentration-dependent phase transition in vitro, resulting in the formation of hydrogels consisting of amyloid-like FUS fibers [30]. This is reminiscent of work on nuclear pore proteins, whose LCS Phenylalanine-Glycine (FG) repeats also form amyloid-like fibers and gels in vitro [31]. Finally, RNA helicases also frequently contain LCS/IDP regions [32], which may facilitate their frequent co-localization with nuclear bodies [33]; This is consistent with the in vivo findings that the LCS domain of helicase Ddx4 drives liquid phase separation in HeLa nuclei [17].…”