Molecular mechanism of selective transport across the Nuclear Pore Complex

Transcription

1 Molecular mechanism of selective transport across the Nuclear Pore Complex David Winogradoff and Aleksei Aksimentiev Physics Department, University of Illinois at Urbana-Champaign May 16, 2017

2 The Nuclear Pore Complex (NPC) The central network of FG-nups is key to passage across the NPC Nuclear pore complexes (NPCs) control traffic across the nuclear envelope. Larger molecules cannot pass through the central network of nucleoporins (nups) without transport factors What is the mechanism of selective transport?

3 SiN Synthetic biomimic of the NPC 20 nm SiN 20 nm 20 nm 20 nm 20 nm Bare pore Bare pore Nup-coated trans d Current (na) G (ns) Conductance B Conductance G (ns) Ac FG-Nups 10 Bare pore Nup-coated Nup-coated Pore (nm) Porediameter diameter d (nm) Voltage (mv) d Figure 1 Biomimetic NPC. a, Side-view schematic showing the device consisting of a 20 nm thin, free-standing silicon nitride window (b Adapted from Kowalczyk, Dekker etis drilled al., using 2011, Nat. Nanotechnol. embedded in a silicon wafer (light green). A nanopore a highly focused electron beam (yellow). b, Sketch showing the exp The biomimetic NPC is engineered by attaching FG-Nupset to aal., solid-state nanopore, and transport of Impb is measured by monitoring th Jovanovic-Talisman, Chait 2009, Nature c, TEM images of the same nanopore with a diameter of 20 nm (top) or 40 nm (bottom) before (left) and after (right) coating with Nup current voltage (I V) curve before (red) and after (blue) coating a 40 nm nanopore with Nup98, showing an increased resistance due to Bare pore G (ns) c b The C. Dekker lab a(tu Delft) developed a minimalistic biomimetic NPC r = 10 nm Bare pore Solid-state pores were coatednup153 with FG-Nups, and a voltage was applied Nup98 across each d (nm) uctance G (ns) 100 d

4 MD simulations of nup NSP1 A - F 2.5 nm Full length NSP1 was split into residue fragments 5 nm x z E y nm z y x 1 nm NSP1 12-residue fragments Each NSP1 system solvated in M KCl Forces keep the protein in the central region; After equilibration, 500mV applied in z 2.5 nm 5 nm 2.5 nm protein content 6 systems of varying NSP1 content (volume fraction) 70% 60% 50% 40% 30% 20%

5 E-field simulations of NSP1 Protein, water, ions, Apply an electric field, E Trace the motion of ions to calculate current I and conductivity σ I(t) = 1 tl NX q i z i time t, desired length l, ion index i, charge q i

6 Nup conductivity from MD and experiment Conductivity from MD Conductance from experiment Conductivity σ (ms/cm) Linear model NSP1 Conductance G (ns) Kowalczyk, Dekker et al., (2011), Nanotechnology. Protein volume fraction (%) Pore diameter d (nm) Protein density (mg/ml) ns MD simulations were performed; 500 mv applied in +z Ion conductivity of NSP1 increases nonlinearly with decreasing protein content Matching experimental results for Nup98 from the Dekker lab

7 Modeling the entire Nuclear Pore Complex 1. The most complete structure 2. Dual resolution MD All-atom Coarse-grained??? Cytoplasmic outer ring inner ring Nuclear outer ring { { { Central channel nups first represented by a coarse-grained (CG) potential Ghavami, Onck et al., 2014, Biophys J. Composite structure developed by Lin, Hoelz et al., Science, 2016

8 Coarse-grained simulations of the NPC Core nups based on (Lin et al., 2016, Science) Central mesh: Onck CG model (Ghavami et al., 2014, Biophys. J.) all-atom nup core smoothed potential C/H = ratio of charged to hydrophobic residues Model tuned to experimentally measured nup sizes (Yamada et al., 2010, Mol. & Cell. Prot.)

9 Coarse-grained simulations of the NPC bonds weakened Time (µs)

10 Density throughout the CG NPC simulation all residues (~53000 total) 10 nm hydrophobic residues (~25% of total)

11 Building an all-atom model of the NPC 1. All-atom model of the core 2. Add the central channel 3. Add the nuclear envelope 4. Solvate the entire system

12 Preliminary estimate of NPC electrical properties protein 1th nanopore z y 2th nth ith y x ith segment (r) = 1 r tanh l R ij = (r)a xy b a Conductance estimated from theory: 65.1 ns R = nx i=1 1 P m j=1 1 R ij I = V/R W. Sei, A. Aksimentiev, submitted Conductance estimated from experiment: [33, 59] ns 1. Bustamante, 1995, J. Membrane Biol. 2. Danker, 1999, PNAS 3. Mazzanti, 2001, Physiol. Rev.

13 Running the all-atom NPC on Blue Waters Running on Blue Waters 50 nodes: 120 ps/day ~100 nm 100 nodes: 260 ps/day 200 nodes: 400 ps/day ~125 nm Entire system: 140 million atoms

14 Conclusion and future directions All-atom MD simulations matched experimental biomimetic results For the 1st time, a complete all-atom model of the NPC was built and run (140 M atoms) The electrical properties of the entire NPC will be characterized The diffusion process of proteins and RNA across the NPC will be modeled

15 Acknowledgements Chris Maffeo Maxim Belkin Chen-Yu Li Aleksei Aksimentiev Wei Si Cees Dekker Ali Ghavami, Patrick Onck