Structural basis for cooperativity of CRM1 export complex formation

Monecke, Thomas; Haselbach, David; Voß, Béla; Russek, Andreas; Neumann, Piotr; Thomson, Emma; Hurt, Ed; Zachariae, Ulrich; Stark, Holger; Grubmüller, Helmut; Dickmanns, Achim; Ficner, Ralf

doi:10.1073/pnas.1215214110

Cited by 80 publications

(113 citation statements)

References 40 publications

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“…These observations fit to the current understanding of export complex formation, in which cargo proteins and Ran•GTP cooperatively bind to CRM1 (see model in Fig. S3B) (18). The binding of Ran•GppNHp to CRM1•Spn1 was largely unaffected by acetylation.…”

Section: Crm1supporting

confidence: 58%

“…In the cytosol, the Importin•Ran•GTP complexes, as well as the ternary exportin•Ran•GTP-cargo complexes, dissociate on binding of RanBP1 and subsequent GTP hydrolysis catalyzed by RanGAP (16,17). The Ran transport cycle closes by translocation of Ran•GDP to the nucleus by the nuclear transport factor 2 (NTF2) (4,(17)(18)(19)(20). Many of these Ran interactions also play important roles in mitotic spindle assembly and nuclear envelope formation.…”

mentioning

confidence: 99%

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Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Boor

Knyphausen

Kuhlmann

et al. 2015

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

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Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.Ran | lysine acetylation | genetic code expansion concept | nucleus | nuclear cytosolic transport T he small GTP-binding protein Ran (Ras-related nuclear) is involved in nucleo-cytoplasmic transport processes, nuclear envelope formation, and the formation of the mitotic spindle (1). Ran, furthermore, has a variety of cytosolic functions and is involved in the cross-talk with the actin cytoskeleton. As a member of the Ras superfamily, Ran is structurally composed of a fold known as the G-domain (GTP-binding domain), a central sixstranded β-sheet that is surrounded by α-helices. Ras-family members bind to GTP and GDP nucleotides with high picomolar affinity. However, only in the GTP-bound form and the switch Iand switch II-loops adopt a stable conformation. Ran has been structurally characterized in great detail, including different nucleotide states and various protein complexes (2-4).In interphase cells, about 90% of cellular Ran is nuclear, and only a minor proportion is cytosolic (5). The localization of the guanine-nucleotide exchange factor (GEF) RCC1 (Regulator of chromosome condensation 1) at the nuclear chromatin and the RanGAP (RanGTPase-activating protein) at the cytosolic site of the nuclear pore creates a gradient of Ran•GTP in the nucleus and Ran•GDP in the cytosol (6-8).In the nucleus, Ran•GTP binds to exportins such as CRM1 (Chromosome region maintenance 1) to transport cargo proteins containing a nuclear export signal (NES) into the cytosol (3, 9, 10). Ran•GTP, furthermore, binds to Importin-β•cargo complexes to release the cargo in the nucleus (11-15). In the cytosol, the Importin•Ran•GTP complexes, as well as the ternary exportin•Ran•GTP-cargo complexes, dissociate on binding of RanBP1 and subsequent GTP hydrolysis catalyzed by RanGAP (16,17). The Ran transport cycle closes by translocation of Ran•GDP to the nucleus by the nuclear transport factor 2 (NTF2) (4,(17)(18)(19)(20). Many of these Ran interactions also ...

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Section: Crm1supporting

confidence: 58%

mentioning

confidence: 99%

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Boor

Knyphausen

Kuhlmann

et al. 2015

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

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“…Cargo binding shifts the equilibrium toward more closed conformations by selecting rather strained conformers of Importin-b from this large pool of different conformations, with the corresponding entropy changes being crucial for the overall thermodynamics of the system. Similar observations were made for the exportin Crm1, which cooperatively adopts more compact conformations upon binding RanGTP and cargo (56). However, available structural models of Importin-b do not fully support this perception.…”

Section: Discussionsupporting

confidence: 59%

MD Simulations and FRET Reveal an Environment-Sensitive Conformational Plasticity of Importin-β

Halder

Dölker

Van

et al. 2015

Biophysical Journal

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The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-β in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Förster resonance energy transfer experiments using dual-fluorophore-labeled Importin-β. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.

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“…3 A and B). In CRM1, the availability of the hydrophobic NES binding cleft is modulated by a RanGTP-dependent rearrangement of an acidic loop (25). The hydrophobic groove observed in ctNup192 NTD is located in a similar position on the outside of the ring, suggesting that the interaction with another nucleoporin at this site is regulated similarly.…”

Section: Resultsmentioning

confidence: 95%

Evidence for an evolutionary relationship between the large adaptor nucleoporin Nup192 and karyopherins

Stuwe

Lin

Collins

et al. 2014

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

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Nucleocytoplasmic transport is facilitated by nuclear pore complexes (NPCs), which are massive proteinaceous transport channels embedded in the nuclear envelope. Nup192 is a major component of an adaptor nucleoporin subcomplex proposed to link the NPC coat with the central transport channel. Here, we present the structure of the ∼110-kDa N-terminal domain (NTD) of Nup192 at 2.7-Å resolution. The structure reveals an open ring-shaped architecture composed of Huntingtin, EF3, PP2A, and TOR1 (HEAT) and Armadillo (ARM) repeats. A comparison of different conformations indicates that the NTD consists of two rigid halves connected by a flexible hinge. Unexpectedly, the two halves of the ring are structurally related to karyopherin-α (Kap-α) and β-karyopherin family members. Biochemically, we identify a conserved patch that binds an unstructured segment in Nup53 and show that a C-terminal tail region binds to a putative helical fragment in Nic96. The Nup53 segment that binds Nup192 is a classical nuclear localization-like sequence that interacts with Kap-α in a mutually exclusive and mechanistically distinct manner. The disruption of the Nup53 and Nic96 binding sites in vivo yields growth and mRNA export defects, revealing their critical role in proper NPC function. Surprisingly, both interactions are dispensable for NPC localization, suggesting that Nup192 possesses another nucleoporin interaction partner. These data indicate that the structured domains in the adaptor nucleoporin complex are held together by peptide interactions that resemble those found in karyopherin•cargo complexes and support the proposal that the adaptor nucleoporins arose from ancestral karyopherins.X-ray crystallography | site-directed mutagenesis | mega assembly | structure-function analysis

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Structural basis for cooperativity of CRM1 export complex formation

Cited by 80 publications

References 40 publications

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

MD Simulations and FRET Reveal an Environment-Sensitive Conformational Plasticity of Importin-β

Evidence for an evolutionary relationship between the large adaptor nucleoporin Nup192 and karyopherins

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