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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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Structure of the RanBP3 RBD.A. Ribbon diagram. The asterisk indicates the surface depression which in homologous RBD structures accommodates the Ran C-helix. B. Structural alignment of the RanBP3 RBD (magenta) with RanBP1 (green). C. Sequence alignment of RBDs of known structure. The β5β6 loop is boxed. Residues in lower case are missing from the structures. RanBP2-1 residues that contact Ran are marked by a circle, triangle or square according to the type of contact (van der Waals, H-bond mediated by a side chain, or H-bond mediated by backbone, respectively) [38]. Marks are coloured according to whether the Ran residue contacted lies in the G-domain (black), effector loop (green), linker (magenta), C-helix (blue) or DEDDDL motif (open triangle).
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pone-0017011-g003: Structure of the RanBP3 RBD.A. Ribbon diagram. The asterisk indicates the surface depression which in homologous RBD structures accommodates the Ran C-helix. B. Structural alignment of the RanBP3 RBD (magenta) with RanBP1 (green). C. Sequence alignment of RBDs of known structure. The β5β6 loop is boxed. Residues in lower case are missing from the structures. RanBP2-1 residues that contact Ran are marked by a circle, triangle or square according to the type of contact (van der Waals, H-bond mediated by a side chain, or H-bond mediated by backbone, respectively) [38]. Marks are coloured according to whether the Ran residue contacted lies in the G-domain (black), effector loop (green), linker (magenta), C-helix (blue) or DEDDDL motif (open triangle).

Mentions: Initial efforts to crystallize the RBD of RanBP3 in complex with Ran were hampered by the weak Ran-binding affinity of this domain, which we estimated by isothermal titration calorimetry (ITC) to correspond to a Kd of 14±0.3 µM (Figure 2; Figure S1 shows the corresponding experiment for full-length RanBP3), in agreement with a previous semi-quantitative study [18]. We therefore pursued the structure of the RBD in its unbound form. We solved the structure at 1.61 Å resolution using experimental phases obtained from a platinum derivative, and at 2.1 Å in a second crystal form by molecular replacement. (Crystallographic statistics are summarized in Table 1). As expected, the RanBP3 RBD adopts a pleckstrin homology fold, composed of 7 anti-parallel β-strands and a C-terminal α-helix. The strands define a continuous sheet with simple up-down topology, forming an imperfect β barrel that juxtaposes strands 4 and 6 and extrudes strand 5 (Figure 3A). The C-terminal helix caps the barrel, packing against strands 1, 2, 5 and 6. The loops at the base of the barrel and a shallow depression on the protein surface between the β1β2 and β5β6 loops (asterisk in Figure 3A and B) correspond to important Ran-binding epitopes in known structures of Ran/RBD complexes. Crystal forms 1 and 2 contain two and four molecules per asymmetric unit, respectively, and aligning these structures reveals variations in the N- and C-terminal residues and in several loops, reflecting the inherent flexibility of these regions (Figure S2; Table S1). In contrast, the β5β6 loop, whose functional role is evoked below, is highly uniform in structure, suggesting a comparatively rigid element. Our crystal structure of the RanBP3 RBD is consistent with an NMR structure determined by a structural genomics consortium [37] (PDB code 2CRF), although aligning the two structures yields a high rmsd value (1.6 Å for 100 Cα residues, omitting variable regions), which we attribute to coordinate errors in the NMR model.


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)

Structure of the RanBP3 RBD.A. Ribbon diagram. The asterisk indicates the surface depression which in homologous RBD structures accommodates the Ran C-helix. B. Structural alignment of the RanBP3 RBD (magenta) with RanBP1 (green). C. Sequence alignment of RBDs of known structure. The β5β6 loop is boxed. Residues in lower case are missing from the structures. RanBP2-1 residues that contact Ran are marked by a circle, triangle or square according to the type of contact (van der Waals, H-bond mediated by a side chain, or H-bond mediated by backbone, respectively) [38]. Marks are coloured according to whether the Ran residue contacted lies in the G-domain (black), effector loop (green), linker (magenta), C-helix (blue) or DEDDDL motif (open triangle).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045386&req=5

pone-0017011-g003: Structure of the RanBP3 RBD.A. Ribbon diagram. The asterisk indicates the surface depression which in homologous RBD structures accommodates the Ran C-helix. B. Structural alignment of the RanBP3 RBD (magenta) with RanBP1 (green). C. Sequence alignment of RBDs of known structure. The β5β6 loop is boxed. Residues in lower case are missing from the structures. RanBP2-1 residues that contact Ran are marked by a circle, triangle or square according to the type of contact (van der Waals, H-bond mediated by a side chain, or H-bond mediated by backbone, respectively) [38]. Marks are coloured according to whether the Ran residue contacted lies in the G-domain (black), effector loop (green), linker (magenta), C-helix (blue) or DEDDDL motif (open triangle).
Mentions: Initial efforts to crystallize the RBD of RanBP3 in complex with Ran were hampered by the weak Ran-binding affinity of this domain, which we estimated by isothermal titration calorimetry (ITC) to correspond to a Kd of 14±0.3 µM (Figure 2; Figure S1 shows the corresponding experiment for full-length RanBP3), in agreement with a previous semi-quantitative study [18]. We therefore pursued the structure of the RBD in its unbound form. We solved the structure at 1.61 Å resolution using experimental phases obtained from a platinum derivative, and at 2.1 Å in a second crystal form by molecular replacement. (Crystallographic statistics are summarized in Table 1). As expected, the RanBP3 RBD adopts a pleckstrin homology fold, composed of 7 anti-parallel β-strands and a C-terminal α-helix. The strands define a continuous sheet with simple up-down topology, forming an imperfect β barrel that juxtaposes strands 4 and 6 and extrudes strand 5 (Figure 3A). The C-terminal helix caps the barrel, packing against strands 1, 2, 5 and 6. The loops at the base of the barrel and a shallow depression on the protein surface between the β1β2 and β5β6 loops (asterisk in Figure 3A and B) correspond to important Ran-binding epitopes in known structures of Ran/RBD complexes. Crystal forms 1 and 2 contain two and four molecules per asymmetric unit, respectively, and aligning these structures reveals variations in the N- and C-terminal residues and in several loops, reflecting the inherent flexibility of these regions (Figure S2; Table S1). In contrast, the β5β6 loop, whose functional role is evoked below, is highly uniform in structure, suggesting a comparatively rigid element. Our crystal structure of the RanBP3 RBD is consistent with an NMR structure determined by a structural genomics consortium [37] (PDB code 2CRF), although aligning the two structures yields a high rmsd value (1.6 Å for 100 Cα residues, omitting variable regions), which we attribute to coordinate errors in the NMR model.

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.

Show MeSH
Related in: MedlinePlus