DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex

Abstract

The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.

Fig. 1

Schematics of DNA origami ring with attached FG-Nups (a), DNA ring (see Supplementary Figure 1 for design details) and one NSP1 protein with a covalently attached oligonucleotide. b Ring with attached NSP1 protein. c DNA ring versions with 8 (left) and 32 (right) NSP1 proteins attached. d DNA ring with 32-NSP1 (left) and 32 mutated NSP1-S (right) attached

Fig. 2

Attachment of NSP1 and NSP1-S to the DNA ring. Top: laser-scanned EtBr images of (a) 1% agarose gel on which DNA origami ring samples were electrophoresed. Middle: same gel scanned in Cy5 excitation/emission channels. Cy5-labeled DNA strands were added to FG-Nup attachments where indicated. Bottom: integrated Cy5 band intensity normalized to EtBr intensity. b Histogram of the number of attached Cy5-labeled oligomers for an 8-attachment ring. Inset shows an exemplary intensity trace of a single-particle recording of a DNA ring with 8 attachment sites incubated with the complementary Cy5-labeled oligonucleotide obtained using total internal reflection microscopy (TIRF) (Methods section and Supplementary Figure 4). See Supplementary Figure 5 for additional intensity traces. c–e Exemplary field-of-view negative staining TEM micrographs of DNA origami NCP-mimic ring without protein, with 32-NSP1, and 32-NSP1-S, respectively. See Supplementary Figure 6 for exemplary particles. Scale bar = 50 nm

Fig. 3

Spatial distribution of FG-Nup densities in DNA rings from cryo-EM and MD simulations. a Schematic representation of (top to bottom): empty ring, 32-NSP1, and 32-NSP1-S. b Corresponding average obtained from aligning and summing individual cryo-EM particles. Number of averaged particles: Ring = 1663, NSP1 = 637, NSP1-S = 1051. See Supplementary Figure 8 for exemplary particles. Scale bar = 50 nm. c, d Time-averaged mass densities of proteins inside the DNA ring obtained from coarse-grained MD simulations averaged in the z-direction, shown in top view (c) and side view (d). e Exemplary snapshot of MD simulations of 32-NSP1 (top) and 32-NSP1-S (bottom), showing that NSP1-S proteins extend further out than NSP1

Fig. 4

Ionic conductance of rings with FG-Nups docked on a solid-state nanopore. a Schematic representation of DNA ring that is docking onto a solid-state nanopore. b Exemplary current (nA) versus voltage (mV) traces for rings without proteins, 8-NSP1, 32-NSP1 and 32-NSP1-S and the bare nanopore. c Exemplary relative conductance (G_ring/G_pore) vs time (s) traces showing the change in conductance upon docking of the ring without protein (gray), 32-NSP1 (yellow), and 32-NSP1-S (blue). d Box plot representation of the relative conductance (G_ring/G_pore) for the empty ring, 8-NSP1 and 32-NSP1. e Same as (d), but for empty ring, 8-NSP1-S and 32-NSP1-S. f Same as (d), but for empty ring, 8-NSP1 and 8-NSP1-S. g Same as (d), but for empty ring, 32-NSP1 and 32-NSP1-S. Each of the panels d–g represents a different nanopore experiment where a series of rings are probed on one particular solid-state nanopore. In the box plot representation in d–g, the blue boxes denote the 25th and 75th percentiles and the red lines represent the median values with the associated wedges representing a 95% confidence interval for the medians (see methods and Supplementary Table 4). h Side view (rz plane) average density distribution for 32-NSP1 placed on a 20 nm-wide nanopore in a 20 nm thin SiN membrane (see Supplementary Figure 14 for an exemplary simulation snapshot and Fig. S15 for density distributions of other variants). i Comparison of experimental reduced conductance values and simulation results (Supplementary Note 2 and Supplementary Tables 5 and 6)