Kap95p Binding Induces the Switch Loops of RanGDP to Adopt the GTP-Bound Conformation: Implications for Nuclear Import Complex Assembly Dynamics Jade K. Forwood 1,2 ⁎†, Thierry G. Lonhienne 2,3 †, Mary Marfori 2 , Gautier Robin 2,4 , Weining Meng 2 , Gregor Guncar 2,4 , Sai M. Liu 5 , Murray Stewart 6 , Bernard J. Carroll 2,3 and Bostjan Kobe 2,4 1 School of Biomedical Sciences, Charles Sturt University, Wagga Wagga 2650, Australia 2 School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia 3 School of Crop, Land and Food Sciences, and ARC Centre of Excellence for Integrative Legume Research, The University of Queensland, Brisbane, Queensland 4072, Australia 4 Institute for Molecular Bioscience and Special Research Centre for Functional and Applied Genomics, The University of Queensland, Brisbane, Queensland 4072, Australia 5 Sir William Dunn School of Pathology, Oxford University, Oxford OX1 3RE, UK 6 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK Received 25 March 2008; received in revised form 31 July 2008; accepted 31 July 2008 Available online 7 August 2008 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-β. Here, we show that RanGDP and importin-β form a stable complex with a micromolar dissociation constant. This complex can be dissociated by importin-β binding partners such as importin-α. 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. © 2008 Elsevier Ltd. All rights reserved. Edited by J. Karn Keywords: nucleocytoplasmic transport; Ran; karyopherin; importin; protein structure *Corresponding author. E-mail address: jforwood@csu.edu.au. † J.K.F. and T.G.L. contributed equally to the work. Abbreviations used: GTP, guanosine 5′-triphosphate; GDP, guanosine 5′-diphosphate; GTPase, guanosine 5′- triphosphatase; GAP, GTPase activating protein; IBB, importin-β-binding; NCS, non-crystallographic symmetry; GST, glutathione-S-transferase; TLS, translation-libration-screw. doi:10.1016/j.jmb.2008.07.090 J. Mol. Biol. (2008) 383, 772–782 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.