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Insights into the function of the CRM1 cofactor RanBP3 from the structure of its Ran-binding domain.

Langer K, Dian C, Rybin V, Müller CW, Petosa C - PLoS ONE (2011)

Bottom Line: RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1.Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex.The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.

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Structural basis of weak Ran-binding.A. Structural alignment of the RanBP3 RBD with the RanBP2-RBD1/Ran complex. Ribbon diagrams show Ran (green), RanBP2-1 (blue) and RanBP3 (magenta). The GTP analog GppNHp is in navy blue. B–D. Details of the interface. The carbon atoms of RanBP2-1 and RanBP3 residues are shown in green and magenta, respectively. B. The Glu→Ser and Lys→Val substitutions within the 1154EWKER/349SWVER motif are predicted to disrupt two salt bridge interactions between Ran and the RBD. C, D. The R1284/S423 (C) and Q1248/G381 (D) substitutions are predicted to disrupt interactions with the Ran 211DEDDDL motif and with Glu158 in the G-domain, respectively. E. RBD surface plots. The two left-hand panels are related to (A) by an approximately 180° rotation about the vertical axis. Top, RanBP2-1 residues within 4 Å of Ran are coloured blue. Bottom, RanBP3 residues identical to RanBP2-1 are coloured magenta. The conserved and contact surfaces are similar in the vicinity of the Ran G-domain and linker (left panels), but not near the Ran C-helix (right panels).
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pone-0017011-g004: Structural basis of weak Ran-binding.A. Structural alignment of the RanBP3 RBD with the RanBP2-RBD1/Ran complex. Ribbon diagrams show Ran (green), RanBP2-1 (blue) and RanBP3 (magenta). The GTP analog GppNHp is in navy blue. B–D. Details of the interface. The carbon atoms of RanBP2-1 and RanBP3 residues are shown in green and magenta, respectively. B. The Glu→Ser and Lys→Val substitutions within the 1154EWKER/349SWVER motif are predicted to disrupt two salt bridge interactions between Ran and the RBD. C, D. The R1284/S423 (C) and Q1248/G381 (D) substitutions are predicted to disrupt interactions with the Ran 211DEDDDL motif and with Glu158 in the G-domain, respectively. E. RBD surface plots. The two left-hand panels are related to (A) by an approximately 180° rotation about the vertical axis. Top, RanBP2-1 residues within 4 Å of Ran are coloured blue. Bottom, RanBP3 residues identical to RanBP2-1 are coloured magenta. The conserved and contact surfaces are similar in the vicinity of the Ran G-domain and linker (left panels), but not near the Ran C-helix (right panels).

Mentions: Structures of a Ran-bound RBD have been determined in the context of three different complexes: the RanBP2-1/Ran, RanBP1/Ran/RanGAP and Yrb1/Ran/CRM1 complexes (Yrb1 is yeast RanBP1) [33], [38], [39]. The Ran-RBD interface is essentially identical in all these structures, as residues mediating Ran recognition are highly conserved across the three RBDs (Figure 3C) and because the interface is unaffected by either CRM1 or RanGAP binding. The Ran-RBD interaction has been described as a “molecular embrace” [38], in which Ran wraps its C-terminal tail around the RBD, and the RBD wraps its N-terminal extension around Ran (Figure 4A). The intermolecular contacts thus fall into three classes: those between the RBD N-terminal extension and the globular Ran guanine-nucleotide binding domain (G domain) (class 1); those between the Ran C-terminal tail and the RBD globular domain (class 2); and those between the two globular domains (class 3). Class 1 contacts are mediated by 8 residues in the RBD N-terminal extension, of which 5 make van der Waals contacts and 3 make H bonds via main chain atoms (Figure 3C). Class 2 contacts involve all three moieties of the Ran C-terminal tail (linker, C-helix, DEDDDL motif) and comprise: H bonds between the Ran linker and RBD strands β3 and β4, van der Waals contacts between the Ran C-helix and the RBD surface depression (asterisk in Figure 3B), and electrostatic interactions between the DEDDDL motif and basic RBD residues. Class 3 includes salt bridge interactions between the EWKER motif in RBD strand β2 and the nucleotide-binding effector loop of Ran.


Insights into the function of the CRM1 cofactor RanBP3 from the structure of its Ran-binding domain.

Langer K, Dian C, Rybin V, Müller CW, Petosa C - PLoS ONE (2011)

Structural basis of weak Ran-binding.A. Structural alignment of the RanBP3 RBD with the RanBP2-RBD1/Ran complex. Ribbon diagrams show Ran (green), RanBP2-1 (blue) and RanBP3 (magenta). The GTP analog GppNHp is in navy blue. B–D. Details of the interface. The carbon atoms of RanBP2-1 and RanBP3 residues are shown in green and magenta, respectively. B. The Glu→Ser and Lys→Val substitutions within the 1154EWKER/349SWVER motif are predicted to disrupt two salt bridge interactions between Ran and the RBD. C, D. The R1284/S423 (C) and Q1248/G381 (D) substitutions are predicted to disrupt interactions with the Ran 211DEDDDL motif and with Glu158 in the G-domain, respectively. E. RBD surface plots. The two left-hand panels are related to (A) by an approximately 180° rotation about the vertical axis. Top, RanBP2-1 residues within 4 Å of Ran are coloured blue. Bottom, RanBP3 residues identical to RanBP2-1 are coloured magenta. The conserved and contact surfaces are similar in the vicinity of the Ran G-domain and linker (left panels), but not near the Ran C-helix (right panels).
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Related In: Results  -  Collection

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pone-0017011-g004: Structural basis of weak Ran-binding.A. Structural alignment of the RanBP3 RBD with the RanBP2-RBD1/Ran complex. Ribbon diagrams show Ran (green), RanBP2-1 (blue) and RanBP3 (magenta). The GTP analog GppNHp is in navy blue. B–D. Details of the interface. The carbon atoms of RanBP2-1 and RanBP3 residues are shown in green and magenta, respectively. B. The Glu→Ser and Lys→Val substitutions within the 1154EWKER/349SWVER motif are predicted to disrupt two salt bridge interactions between Ran and the RBD. C, D. The R1284/S423 (C) and Q1248/G381 (D) substitutions are predicted to disrupt interactions with the Ran 211DEDDDL motif and with Glu158 in the G-domain, respectively. E. RBD surface plots. The two left-hand panels are related to (A) by an approximately 180° rotation about the vertical axis. Top, RanBP2-1 residues within 4 Å of Ran are coloured blue. Bottom, RanBP3 residues identical to RanBP2-1 are coloured magenta. The conserved and contact surfaces are similar in the vicinity of the Ran G-domain and linker (left panels), but not near the Ran C-helix (right panels).
Mentions: Structures of a Ran-bound RBD have been determined in the context of three different complexes: the RanBP2-1/Ran, RanBP1/Ran/RanGAP and Yrb1/Ran/CRM1 complexes (Yrb1 is yeast RanBP1) [33], [38], [39]. The Ran-RBD interface is essentially identical in all these structures, as residues mediating Ran recognition are highly conserved across the three RBDs (Figure 3C) and because the interface is unaffected by either CRM1 or RanGAP binding. The Ran-RBD interaction has been described as a “molecular embrace” [38], in which Ran wraps its C-terminal tail around the RBD, and the RBD wraps its N-terminal extension around Ran (Figure 4A). The intermolecular contacts thus fall into three classes: those between the RBD N-terminal extension and the globular Ran guanine-nucleotide binding domain (G domain) (class 1); those between the Ran C-terminal tail and the RBD globular domain (class 2); and those between the two globular domains (class 3). Class 1 contacts are mediated by 8 residues in the RBD N-terminal extension, of which 5 make van der Waals contacts and 3 make H bonds via main chain atoms (Figure 3C). Class 2 contacts involve all three moieties of the Ran C-terminal tail (linker, C-helix, DEDDDL motif) and comprise: H bonds between the Ran linker and RBD strands β3 and β4, van der Waals contacts between the Ran C-helix and the RBD surface depression (asterisk in Figure 3B), and electrostatic interactions between the DEDDDL motif and basic RBD residues. Class 3 includes salt bridge interactions between the EWKER motif in RBD strand β2 and the nucleotide-binding effector loop of Ran.

Bottom Line: RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1.Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex.The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.

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