Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex.
Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.
Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, San Francisco, CA 94143; Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94143; California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA 94143;
Barak
B
Raveh
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461;
Jerome M
JM
Karp
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461;
Samuel
S
Sparks
NMR Department, New York Structural Biology Center, New York, NY, 10027;
Kaushik
0000-0002-9653-3842
K
Dutta
Laboratory of Cellular and Structural Biology, Rockefeller University, New York, NY 10065.
Michael P
MP
Rout
Department of Bioengineering and Therapeutic Sciences, University of California at San Francisco, San Francisco, CA 94143; Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94143; California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA 94143; david.cowburn@einstein.yu.edu sali@salilab.org.
Andrej
A
Sali
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461; Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461; david.cowburn@einstein.yu.edu sali@salilab.org.
David
D
Cowburn
D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D003198:Computer Simulation; D005998:Glycine / Q000737:chemistry*; D008954:Models, Biological; D008956:Models, Chemical; D056004:Molecular Dynamics Simulation; D022022:Nuclear Pore / Q000737:chemistry* / Q000648:ultrastructure*; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains; D020449:Repetitive Sequences, Amino Acid
D010649:Phenylalanine; D005998:Glycine
NMR; molecular dynamics; nuclear pore; nucleoporins; transport factors