PDBsum entry 3ea5

Transport protein

PDB id: 3ea5

Contents

Protein chains

174 a.a. *

859 a.a. *

Ligands

GDP ×2

Metals

_MG ×4

Waters ×364

* Residue conservation analysis

PDB id:

3ea5

Name:

Transport protein

Title:

Kap95p binding induces the switch loops of rangdp to adopt the gtp- bound conformation: implications for nuclear import complex assembly dynamics

Structure:

Gtp-binding nuclear protein ran. Chain: a, c. Synonym: gtpase ran, ras-related nuclear protein, ras-like protein tc4, androgen receptor-associated protein 24. Engineered: yes. Mutation: yes. Importin subunit beta-1. Chain: b, d. Synonym: karyopherin subunit beta-1, karyopherin-95, importin-95.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: ran, ara24. Expressed in: escherichia coli. Expression_system_taxid: 562. Saccharomyces cerevisiae. Brewer's yeast,lager beer yeast,yeast. Organism_taxid: 4932.

Resolution:

2.50Å R-factor: 0.192 R-free: 0.232

Authors:

J.K.Forwood,J.K.Lonhienne,G.Guncar,M.Stewart,M.Marfori,B.Kobe

Key ref:

J.K.Forwood et al. (2008). Kap95p binding induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics. J Mol Biol, 383, 772-782. PubMed id: 18708071 DOI: 10.1016/j.jmb.2008.07.090

Date:

24-Aug-08 Release date: 21-Oct-08

Protein chains

P62826  (RAN_HUMAN) -  GTP-binding nuclear protein Ran from Homo sapiens

Seq: 216 a.a.

Struc: 174 a.a.

Protein chains

Q06142  (IMB1_YEAST) -  Importin subunit beta-1 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 861 a.a.

Struc: 859 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains A, C: E.C.3.6.5.- - ?????

DOI no: 10.1016/j.jmb.2008.07.090

J Mol Biol 383:772-782 (2008)

PubMed id: 18708071

Kap95p binding induces the switch loops of RanGDP to adopt the GTP-bound conformation: implications for nuclear import complex assembly dynamics.

J.K.Forwood, T.G.Lonhienne, M.Marfori, G.Robin, W.Meng, G.Guncar, S.M.Liu, M.Stewart, B.J.Carroll, B.Kobe.

ABSTRACT

The asymmetric distribution of the nucleotide-bound state of Ran across the nuclear envelope is crucial for determining the directionality of nuclear transport. In the nucleus, Ran is primarily in the guanosine 5'-triphosphate (GTP)-bound state, whereas in the cytoplasm, Ran is primarily guanosine 5'-diphosphate (GDP)-bound. Conformational changes within the Ran switch I and switch II loops are thought to modulate its affinity for importin-beta. Here, we show that RanGDP and importin-beta form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-beta binding partners such as importin-alpha. Surprisingly, the crystal structure of the Kap95p-RanGDP complex shows that Kap95p induces the switch I and II regions of RanGDP to adopt a conformation that resembles that of the GTP-bound form. The structure of the complex provides insights into the structural basis for the gradation of affinities regulating nuclear protein transport.

Selected figure(s)

Figure 2.
Fig. 2. Nucleotide-bound state of Ran. (a) Identification of nucleotide bound to Ran by reverse-phase HPLC confirmed the presence of GDP. The stoichiometry of bound nucleotide was assessed by titrating GDP and GTP standards (1.1, 0.58, 0.29, 0.14 nmol). This revealed that 0.71 nmol GDP bound to 0.86 nmol Ran, which is consistent with 1:1 stoichiometry; GTP was not detected. (b) Annealed omit F[o] − F[c] map near the nucleotide bound to Ran (chain A) contoured at 5.0σ (left), and anomalous map (right, contoured at 3.5σ) with a GDP molecule and magnesium ion superimposed.

Figure 3.
Fig. 3. Structure of Kap95p–RanGDP complex. (a) Two views of the Kap95p–RanGDP structure, related by a 90° rotation around the x-axis. Kap95p is shown in cyan, Ran is shown in pink, and the basic patch region is highlighted in red. (b) Superposition of free RanGDP (PDB ID 1BYU^18) and RanGDP in complex with Kap95p. Switch regions are highlighted in light blue (switch I), magenta (switch II), and pink (basic patch) for free RanGDP, and in blue (switch I), purple (switch II), and red (basic patch) for RanGDP in complex with Kap95p. (c) Superposition of RanGDP and RanGTP (PDB ID 2BKU^5) in complex with Kap95p; RanGDP switch regions are blue (switch I), purple (switch II), and red (basic patch); RanGTP switch regions are light blue (switch I), magenta (switch II), and pink (basic patch). (d) Structural comparison of Kap95p–RanGTP,^5 Kap95p–RanGDP, and HEAT repeats 1–11 of importin-β in complex with PTHrP.^27 The schematic diagrams summarize the conformational differences. The conformational shift in the basic patch region of Ran exposes a part of Kap95p HEAT repeat 7 that is important for binding NLS-containing importin-β cargoes including PTHrP,^27 SREBP-2,^28 and importin α.^12 It is unlikely that the observed structural differences between Kap95p–RanGTP and Kap95p–RanGDP are the result of crystal packing, because they both possessed an equivalent arrangement of the protein molecules.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 383, 772-782) copyright 2008.

Figures were selected by an automated process.