Molecular basis of C9orf72 poly-PR interference with the β-karyopherin family of nuclear transport receptors

Abstract

Nucleocytoplasmic transport (NCT) is affected in several neurodegenerative diseases including C9orf72-ALS. It has recently been found that arginine-containing dipeptide repeat proteins (R-DPRs), translated from C9orf72 repeat expansions, directly bind to several importins. To gain insight into how this can affect nucleocytoplasmic transport, we use coarse-grained molecular dynamics simulations to study the molecular interaction of poly-PR, the most toxic DPR, with several Kapβs (importins and exportins). We show that poly-PR Kapβ binding depends on the net charge per residue (NCPR) of the Kapβ, salt concentration of the solvent, and poly-PR length. Poly-PR makes contact with the inner surface of most importins, which strongly interferes with Kapβ binding to cargo-NLS, IBB, and RanGTP in a poly-PR length-dependent manner. Longer poly-PRs at higher concentrations are also able to make contact with the outer surface of importins that contain several binding sites to FG-Nups. We also show that poly-PR binds to exportins, especially at lower salt concentrations, interacting with several RanGTP and FG-Nup binding sites. Overall, our results suggest that poly-PR might cause length-dependent defects in cargo loading, cargo release, Kapβ transport and Ran gradient across the nuclear envelope.

Liquid-liquid phase separation of intrinsically disordered FG-Nups is driven by highly-dynamic hydrophobic FG-motifs

“…All coarse-grained MD simulations are done with our 1-BPA model for intrinsically disordered proteins. This implicit-solvent model at amino-acid resolution samples from realistic backbone conformations and accounts for hydrophobic, electrostatic and cation–π interactions [38–40, 51]. Several hydrophobic interactions are fine-tuned to better match the experimental Stokes radii of yeast FG-Nup segments.…”

“…However, a complete assay evaluating the LLPS characteristics for all yeast FG-Nups and their different domains is missing. Here, we investigate LLPS of FG-Nups by carrying out coarse-grained molecular dynamics (CGMD) simulations using a one-bead-per-amino-acid (1-BPA) MD model [38][39][40]. This model has been used previously to study nuclear transport through the yeast NPC [39,[41][42][43][44][45][46] and biomimetic nanopores [47][48][49][50], and to analyze the LLPS of dipeptide repeat proteins [51].…”