Molecular interactions of FG nucleoporin repeats at high resolution

Abstract

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient β-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG–FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities.

Visualizing the disordered nuclear transport machinery in situ

“…In reconstituted condensates, NUP98 FG domains tend to gelate over time, potentially by forming β-structures 26 , 51 , 52 . During the required FLIM–FRET measurement time of approximately 5 min, the resulting distance could thus be affected by condensate ageing despite using freshly prepared FG droplets.…”

Improving the hole picture: towards a consensus on the mechanism of nuclear transport

“… Because of their tethering, and lack of tertiary protein structure, the FG Nups are in a polymer brush formation. Controversy centers on whether the brush is entirely intrinsically disordered [ 65 , 66 ], with the degree and speed of motion varying with distance from the tether, and the grafting density (upper) or whether cohesion of the FG repeats results in varying degrees of condensation leading to gels [ 73 , 74 ] (middle) or amyloids [ 75 , 76 ] (lower). …”

“…Based on observations of apparent radius of gyration [ 36 ], the GLFG flavor of FG repeats (above) has generally been assigned a higher degree of cohesiveness. In solution this is reflected by GLFG repeats being able to display the complete range of polymorphisms (above) [ 73 , 75 , 76 , 88 ]. There is no direct evidence testing which of these states or combination of states is present in the NPC, even though the suggestion that a high degree of condensation (or cohesion) plays a functional role has been widely propagated [ 74 , 75 , 81 , 88–91 ].…”

Coordinating nucleoporin condensation and nuclear pore complex assembly

“…Thus, mechanisms must presumably be in place to prevent the premature assembly of larger elements of the NPC structure, which would be an impediment to nuclear import given the size constraints for nuclear transport [54–57]. Equally important, the finding that purified FG‐Nups form condensates that can mature into gels, aggregates, and in some cases amyloids (depending on amino acid composition) [12,13,28,58], suggests that these aggregates or amyloids need to be counteracted by molecular chaperones or other suitable machinery to keep FG‐Nups in a transport‐competent state. Upon arrival in the nuclear compartment, however, such factors need to dissociate to enable staged NPC assembly in juxtaposition to the INM (Fig.…”