Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export

Port, Sarah A.; Monecke, Thomas; Dickmanns, Achim; Spillner, Christiane; Hofele, Romina V.; Urlaub, Henning; Ficner, Ralf; Kehlenbach, Ralph H.

doi:10.1016/j.celrep.2015.09.042

Cited by 88 publications

(142 citation statements)

References 48 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…8A). Thus, as previously pointed out (Port et al 2015), the FG repeat binding sites of CRM1 (Xpo1p) appear to be highly conserved from yeast to humans. 8B).…”

Section: Conservation Of the Fg Repeat Binding Sites On Crm1 (Xpo1p) mentioning

confidence: 55%

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p

et al. 2017

View full text Add to dashboard Cite

Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus.

show abstract

“…8A). Thus, as previously pointed out (Port et al 2015), the FG repeat binding sites of CRM1 (Xpo1p) appear to be highly conserved from yeast to humans. 8B).…”

Section: Conservation Of the Fg Repeat Binding Sites On Crm1 (Xpo1p) mentioning

confidence: 55%

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p

et al. 2017

View full text Add to dashboard Cite

show abstract

“…In both cases, the NES binding pocket of CRM1 is occupied or blocked and suggests that an open conformation could be necessary for import of the free CRM1. While crystalographic analysis of the trimeric export complex bound to an FG repeat peptide identified three domains on CRM1 that form contacts with the FG repeats, none of them are coincident with the NES binding pocket [26,27]. However, binding of LMB or NES cargo to CRM1 does induce conformational changes that could alter interactions with the NPC, to prevent nuclear import of the LMB-CRM1 or high affinity NES-CRM1 proteins.…”

Section: Discussionmentioning

confidence: 99%

Leptomycin B alters the subcellular distribution of CRM1 (Exportin 1)

Rahmani

Dean

2017

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

CRM1 (chromosome maintenance region 1, Exportin 1) binds to nuclear export signals and is required for nucleocytoplasmic transport of a large variety of proteins and RNP complexes. Leptomycin B (LMB), the first specific inhibitor of CRM1 identified, binds covalently to cysteine 528 in the nuclear export signal binding region of CRM1 leading to the inhibition of protein nuclear export. Although the biochemical mechanisms of action of CRM1 inhibitors such as LMB are well studied, the subcellular effects of inhibition on CRM1 are unknown. We have found that LMB causes CRM1 to redistribute from the nucleus to the cytoplasm in A549 cells. A significant decrease in nuclear CRM1 coupled with an increase in cytoplasmic CRM1 was sustained for up to 4 hours, while there was no change in total CRM1 protein in fractionated cells. Cells expressing an LMB insensitive HA-tagged CRM1-C528S protein were unaffected by LMB treatment, whereas HA-tagged wildtype CRM1 redistributed from the nucleus to the cytoplasm with LMB treatment, similar to endogenous CRM1. GFP-tagged CRM1 protein microinjected into the cytoplasm of A549 cells distributed throughout the cell in untreated cells remained primarily cytoplasmic in LMB-treated cells. Upon nuclear microinjection, GFP-CRM1 translocated to and accumulated in the cytoplasm of LMB-treated cells. Thus, LMB binds to CRM1 and causes its redistribution to the cytoplasm by inhibiting its nuclear import. Decreasing the nuclear availability of CRM1 likely contributes to the accumulation of CRM1 cargo proteins in the nucleus, suggesting a new mechanism of action for LMB.

show abstract

“…TFs, such as Kap95 and NTF2, were previously suggested to use multiple interaction sites for FG motifs based on NMR chemical shift analysis (34) and simulation (33). Most recently, the export factor Crm1 was crystallized with eight simultaneously bound FG motifs from three regions of the FG Nup Nup214 (56). Indeed, in both the TIP4P-D and TIP4P-Ew simulations, the FSFG motifs formed multivalent contacts at many different sites (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Raveh

Karp

Sparks

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

131

View full text Add to dashboard Cite

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.nuclear pore | molecular dynamics | nucleoporins | transport factors | NMR T he nuclear pore complex (NPC) regulates macromolecular transport between the nucleus and the cytoplasm in all eukaryotic cells (1, 2). The NPC allows for passive diffusion of small molecules including sugars, ions, and water, while simultaneously imposing size-dependent exclusion of macromolecules without nuclear transport factor (TF) binding sites, generating unique nuclear and cytoplasmic compartments (3, 4). Larger macromolecules can bypass the selectivity barrier of the NPC by interacting with TFs that shuttle cargo through the central channel of the pore. In pathological conditions, including many cancers and viral infections, the NPC is hijacked and used to transport undesired particles, or it is modified to attenuate the cellular responses to disease onset (5, 6).TFs traverse the NPC by selective and reversible association with disordered phenylalanine-glycine (FG) repeat domains of the FG nucleoporin proteins (FG Nups), which line the surface of the NPC (7-14). Each FG repeat domain consists of 5-50 FG repeats. In turn, each FG repeat consists of a single FG motif of various sequence flavors, such as the FSFG and GLFG motifs, and 10-30 spacer residues that separate consecutive FG motifs (15, 16). The spacer residues are strongly hydrophilic (17) and rich ...

show abstract

Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export

Cited by 88 publications

References 48 publications

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p

Leptomycin B alters the subcellular distribution of CRM1 (Exportin 1)

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Contact Info

Product

Resources

About