Abstract
Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP−CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an α-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.
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Acknowledgements
We thank A. Berndt and A. Dickmanns for performing the final purification step and the crystallization of the RanGTP–CRM1 complex, T.A. Rapoport, S. Frey, S. Güttler, C. Siebenhaar and B. Hülsmann for critical reading of the manuscript, B. Hülsmann for preparing the Xenopus egg extract, the Bavarian NMR Centre (BNMRZ) for NMR measurement time, the staff of synchrotron beamlines at the Swiss Light Source (SLS, Villigen, X10SA, PXII) and the European Synchrotron Radiation Facility (ESRF, Grenoble, ID 23-2) for assistance during data collection, and M. Wahl, V. Pena and J. Wawrzinek for setting up the crystallization facility at the MPI. This study was funded by the Max-Planck-Gesellschaft, the Boehringer Ingelheim Fonds and the Alfried Krupp von Bohlen und Halbach Foundation (fellowships to T.G.), EMBO (fellowship to T. Madl), the Austrian Science Fund (FWF, Schrödinger fellowship to T. Madl), European Commission contract 3D Repertoire (LSHG-CT-2005-512028 to M.S.) and contract EU-NMR (No. RII3-026145).
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T.G. conceived biochemical and crystallization experiments, prepared protein constructs (with the help of D.D.), performed the biochemical experiments and export assays, crystallized the export complexes, contributed to crystallographic data collection and structure refinement, prepared the samples for the NMR study, interpreted data and contributed to the writing of the manuscript (including the figures). T. Madl conceived and performed NMR experiments, interpreted the NMR data and contributed to the writing of the manuscript (including the NMR figures). L.C. conducted initial NMR experiments. P.N. performed crystallographic data collection, data processing and structure refinement, and contributed to the interpretation of the crystallographic data. T. Monecke carried out crystallographic data collection. R.F. conducted molecular replacement for the binary CRM1–RanGTP complex. M.S. conceived NMR experiments, interpreted NMR data and contributed to the writing of the manuscript. D.G. conceived biochemical and crystallization experiments, prepared home-made affinity matrices, interpreted data and wrote the manuscript. All authors commented on the paper.
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Güttler, T., Madl, T., Neumann, P. et al. NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nat Struct Mol Biol 17, 1367–1376 (2010). https://doi.org/10.1038/nsmb.1931
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DOI: https://doi.org/10.1038/nsmb.1931
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