Architecture of CRM1/Exportin1 suggests how cooperativity is achieved during formation of a nuclear export complex.

CRM1/Exportin1 mediates the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES) by forming a cooperative ternary complex with the NES-bearing substrate and the small GTPase Ran. We present a structural model of human CRM1 based on a combination of X-ray crystallography, homology modeling, and electron microscopy. The architecture of CRM1 resembles that of the import receptor transportin1, with 19 HEAT repeats and a large loop implicated in Ran binding. Residues critical for NES recognition are identified adjacent to the cysteine residue targeted by leptomycin B (LMB), a specific CRM1 inhibitor. We present evidence that a conformational change of the Ran binding loop accounts for the cooperativity of Ran- and substrate binding and for the selective enhancement of CRM1-mediated export by the cofactor RanBP3. Our findings indicate that a single architectural and mechanistic framework can explain the divergent effects of RanGTP on substrate binding by many import and export receptors.

[1]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[2]  E. Wright,et al.  Structure of functional single AQP0 channels in phospholipid membranes. , 2003, Journal of molecular biology.

[3]  B. Paschal,et al.  A cytosolic activity distinct from crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Utz Fischer,et al.  The HIV-1 Rev Activation Domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs , 1995, Cell.

[5]  P. Chacón,et al.  Multi-resolution contour-based fitting of macromolecular structures. , 2002, Journal of molecular biology.

[6]  Karsten Weis,et al.  Regulating Access to the Genome Nucleocytoplasmic Transport throughout the Cell Cycle , 2003, Cell.

[7]  F. Bischoff,et al.  CRM1-mediated Recycling of Snurportin 1 to the Cytoplasm , 1999, The Journal of cell biology.

[8]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[9]  G. Blobel,et al.  Karyopherins and nuclear import. , 2001, Current opinion in structural biology.

[10]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[11]  M. Fornerod,et al.  The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88 , 1997, The EMBO journal.

[12]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[13]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[14]  George M Sheldrick,et al.  Substructure solution with SHELXD. , 2002, Acta crystallographica. Section D, Biological crystallography.

[15]  Y. Wang,et al.  Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. , 1997, Chemistry & biology.

[16]  Alfred Wittinghofer,et al.  Structural View of the Ran–Importin β Interaction at 2.3 Å Resolution , 1999, Cell.

[17]  Helen R Saibil,et al.  The Chaperonin ATPase Cycle: Mechanism of Allosteric Switching and Movements of Substrate-Binding Domains in GroEL , 1996, Cell.

[18]  Thomas L. Madden,et al.  BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. , 1999, FEMS microbiology letters.

[19]  Minoru Yoshida,et al.  CRM1 Is an Export Receptor for Leucine-Rich Nuclear Export Signals , 1997, Cell.

[20]  F. Bischoff,et al.  RanBP3 influences interactions between CRM1 and its nuclear protein export substrates , 2001, EMBO reports.

[21]  P. Bork,et al.  A Novel Class of RanGTP Binding Proteins , 1997, The Journal of cell biology.

[22]  C. Dargemont,et al.  Evidence for a role of CRM1 in signal-mediated nuclear protein export. , 1997, Science.

[23]  C. Chothia,et al.  Intermediate sequences increase the detection of homology between sequences. , 1997, Journal of molecular biology.

[24]  D. Fujiwara,et al.  Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. , 1994, The Journal of biological chemistry.

[25]  D. L. Weeks,et al.  RanGTP-Regulated Interactions of CRM1 with Nucleoporins and a Shuttling DEAD-Box Helicase , 1999, Molecular and Cellular Biology.

[26]  G. Wahl,et al.  A leucine‐rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking , 1999, The EMBO journal.

[27]  G D Schuler,et al.  A workbench for multiple alignment construction and analysis , 1991, Proteins.

[28]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[29]  U. Kutay,et al.  Nucleocytoplasmic transport: taking an inventory , 2003, Cellular and Molecular Life Sciences CMLS.

[30]  C. Dargemont,et al.  Domains of Crm1 involved in the formation of the Crm1, RanGTP, and leucine-rich nuclear export sequences trimeric complex. , 1999, Experimental cell research.

[31]  S. Eddy.  Hidden Markov models. , 1996, Current opinion in structural biology.

[32]  Benjamin A. Shoemaker,et al.  CDD: a database of conserved domain alignments with links to domain three-dimensional structure , 2002, Nucleic Acids Res..

[33]  Angela Bachi,et al.  PHAX, a Mediator of U snRNA Nuclear Export Whose Activity Is Regulated by Phosphorylation , 2000, Cell.

[34]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[35]  Y. Hakata,et al.  A Multifunctional Domain in Human CRM1 (Exportin 1) Mediates RanBP3 Binding and Multimerization of Human T-Cell Leukemia Virus Type 1 Rex Protein , 2003, Molecular and Cellular Biology.

[36]  Richard Bayliss,et al.  Structural basis for the interaction between NTF2 and nucleoporin FxFG repeats , 2002, The EMBO journal.

[37]  J. Conway,et al.  Methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution. , 1999, Journal of structural biology.

[38]  M. Fitzgibbon,et al.  Structure of Mitogen-activated Protein Kinase-activated Protein (MAPKAP) Kinase 2 Suggests a Bifunctional Switch That Couples Kinase Activation with Nuclear Export* , 2002, The Journal of Biological Chemistry.

[39]  G Vriend,et al.  WHAT IF: a molecular modeling and drug design program. , 1990, Journal of molecular graphics.

[40]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification , 2022 .

[41]  Roger Y Tsien,et al.  Identification of a signal for rapid export of proteins from the nucleus , 1995, Cell.

[42]  Minoru Yoshida,et al.  CRM1 is responsible for intracellular transport mediated by the nuclear export signal , 1997, Nature.

[43]  G. Ghosh,et al.  The Crystal Structure of the IκBα/NF-κB Complex Reveals Mechanisms of NF-κB Inactivation , 1998, Cell.

[44]  Peer Bork,et al.  HEAT repeats in the Huntington's disease protein , 1995, Nature Genetics.

[45]  Søren Brunak,et al.  NESbase version 1.0: a database of nuclear export signals , 2003, Nucleic Acids Res..

[46]  N. Pavletich,et al.  Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms , 1995, Science.

[47]  A. Joachimiak,et al.  Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae. , 2000, Journal of molecular biology.

[48]  Richard Bayliss,et al.  Structural Basis for the Interaction between FxFG Nucleoporin Repeats and Importin-β in Nuclear Trafficking , 2000, Cell.

[49]  G. Bricogne,et al.  [27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. , 1997, Methods in enzymology.

[50]  G. Cingolani,et al.  Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta. , 2002, Molecular cell.

[51]  W. Kabsch,et al.  Atomic structure of the actin: DNase I complex , 1990, Nature.

[52]  Karsten Weis,et al.  Exportin 1 (Crm1p) Is an Essential Nuclear Export Factor , 1997, Cell.

[53]  C. Müller,et al.  Structure of importin-β bound to the IBB domain of importin-α , 1999, Nature.

[54]  G. Blobel,et al.  Uncoupling Kapβ2 Substrate Dissociation and Ran Binding , 2002 .

[55]  M. Lindsay,et al.  Ran-Binding Protein 3 Is a Cofactor for Crm1-Mediated Nuclear Protein Export , 2001, The Journal of cell biology.

[56]  M. Yaffe,et al.  Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding. , 1999, Molecular cell.

[57]  G. Blobel,et al.  Structure of the nuclear transport complex karyopherin-β2–Ran˙GppNHp , 1999, Nature.

[58]  N. Kudo,et al.  Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[59]  E. Yamashita,et al.  The Structure of Importin-ß Bound to SREBP-2: Nuclear Import of a Transcription Factor , 2003, Science.

[60]  D. Svergun,et al.  Conformational Variability of Nucleo-cytoplasmic Transport Factors* , 2004, Journal of Biological Chemistry.

[61]  P Bork,et al.  Comparison of ARM and HEAT protein repeats. , 2001, Journal of molecular biology.