Rapid Brownian Motion Primes Ultrafast Reconstruction of Intrinsically Disordered Phe-Gly Repeats Inside the Nuclear Pore Complex

Abstract

Conformational behavior of intrinsically disordered proteins, such as Phe-Gly repeat domains, alters drastically when they are confined in, and tethered to, nan channels. This has challenged our understanding of how they serve to selectively facilitate translocation of nuclear transport receptor (NTR)-bearing macromolecules. Heterogeneous FG-repeats, tethered to the NPC interior, nonuniformly fill the channel in a diameter-dependent manner and adopt a rapid Brownian motion, thereby forming a porous and highly dynamic polymeric meshwork that percolates in radial and axial directions and features two distinguishable zones: a dense hydrophobic rod-like zone located in the center, and a peripheral low-density shell-like zone. The FG-meshwork is locally disrupted upon interacting with NTR-bearing macromolecules, but immediately reconstructs itself between 0.44 μs and 7.0 μs, depending on cargo size and shape. This confers a perpetually-sealed state to the NPC, and is solely due to rapid Brownian motion of FG-repeats, not FG-repeat hydrophobic bonds. Elongated-shaped macromolecules, both in the presence and absence of NTRs, penetrate more readily into the FG-meshwork compared to their globular counterparts of identical volume and surface chemistry, highlighting the importance of the shape effects in nucleocytoplasmic transport. These results can help our understanding of geometrical effects in, and the design of, intelligent and responsive biopolymer-based materials in nanofiltration and artificial nanopores.

Figure 1. Central channel with all FG-repeats after equilibration.

The wall is shown as semi-transparent. Different monomers in FG-domains are colored according to their properties (see SI for details) as following: brown is hydrophobic (HB), yellow is hydrophobic-negative (HN), cyan is hydrophobic-positive (HP), red is purely negative (PN), blue is purely positive (PP), and white is hydrophilic with zero net charge (HL). (A) Side view. A part of the wall is cut for a better representation of inside the channel. (B) Top view.

Figure 2. Radial concentration of FG-repeats inside the NPC channel.

(A) Time-dependent concentration fluctuations in radial direction at five different radii inside the channel. At each R_i, the concentration is calculated within a cylindrical shell (the ring inside the yellow circle) with a width of the monomer’s vdW diameter. (B) Time-averaged 2D map of radial concentration of all AAs within the FG-repeat domains. The concentrations are averaged along the channel main axis over 20 ms. The diameter of the rod-like high-density zone is about 22 nm.

Figure 3. The 3D spatial density map of inter- and intra-FG-repeats hydrophobic interactions inside the channel.

We pinpointed every single hydrophobic interaction and recorded its coordinates along with the strength of interaction throughout the simulation. Each dim point shows a single hydrophobic interaction, and thus, the color intensity is proportional to the number of hydrophobic interactions. The channel is represented as a wired frame for clarity. (A) side view and (B) top view snapshots.

Figure 4. Reconstruction pattern of the FG-meshwork after a 20-nm globular macromolecule passes through.

Black dots are simulation data and red line shows the fitted eq. (1). Inset: the reconstruction pattern of the FG-meshwork for an equivalent elongated macromolecule (with the diameter of 11.6 nm and the length of 40 nm).

Figure 5. Distribution of the reconstruction characteristic time for different the cavity size of globular and elongated NTR-bearing macromolecules.

Inset: The time of local diffusional motion versus the particle diameter for a globular particles diffusing in cytoplasmic viscosity.

Figure 6. The rapid and reversible collapse of individual FG-repeats in the mid-channel as the NTR-bearing macromolecule approaches from the top.

Left graph shows the brush height as a function of approaching distance. Right graph represents after the macromolecule is removed how fast FG-repeats regain their heights as a function of time.

Figure 7. The side-view of the yeast NPC central channel based on the prediction of the current study, representing two zones inside the FG-meshwork (Fig. 2).

Dashed lines are to guide the eye to distinguish between two zones.