Intramolecular Cohesion of Coils Mediated by Phenylalanine–Glycine Motifs in the Natively Unfolded Domain of a Nucleoporin

V V Krishnan,Michael E Colvin,Justin Yamada,Michael F Rexach,Samir S Patel,Daniel P Denning,Edmond Y Lau,Philip E Bourne

doi:10.1371/journal.pcbi.1000145

Abstract

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine–glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin–cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

Highlights

The nuclear pore complex is a supramolecular protein structure in the nuclear envelope that controls nucleo-cytoplasmic traffic and communication (Figure 1A) [1]
In the analysis that follows we first used molecular dynamics (MD) simulations to generate a statistical ensemble of coil conformations for a 111 amino acid (AA) region of the Nup116 FG domain containing ten FG motifs, and of a mutant version thereof lacking the phenylalanines in the ten FG motifs (F.A mutant) (Figure 1B and 1C)
The simulated FG domains were expressed in bacteria, purified to homogeneity, and analyzed by nuclear magnetic resonance (NMR) spectroscopy and sizing columns to quantify their average shape through measurements of diffusion coefficient and Stokes radii

Summary

Introduction

The nuclear pore complex is a supramolecular protein structure in the nuclear envelope that controls nucleo-cytoplasmic traffic and communication (Figure 1A) [1]. A group that contains numerous phenylalanineglycine repeats (FG nups) (a subset is shown in Figure 1B) line the transport conduit of the NPC (Figure 1A) These FG nups function as stepping-stones for karyopherin movement across the NPC [5,6] and as structural elements of the NPC protein diffusion barrier [7,8]. Since there are ,150 FG nups in each NPC [4], it is currently hypothesized that its transport conduit is lined and/or flanked by 150 natively unfolded FG domains. Together these FG domains constitute ,12% of the total NPC mass or .6.5 MDa of its ,55 MDa structure in yeast [10].

Methods

Results

Conclusion