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      Electrostatic Interactions Involving the Extreme C Terminus of Nuclear Export Factor CRM1 Modulate Its Affinity for Cargo*

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          Abstract

          The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A) α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus.

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          The nuclear pore complex: bridging nuclear transport and gene regulation.

          Although the nuclear pore complex (NPC) is best known for its primary function as the key regulator of molecular traffic between the cytoplasm and the nucleus, a growing body of experimental evidence suggests that this structure participates in a considerably broader range of cellular activities on both sides of the nuclear envelope. Indeed, the NPC is emerging as an important regulator of gene expression through its influence on the internal architectural organization of the nucleus and its apparently extensive involvement in coordinating the seamless delivery of genetic information to the cytoplasmic protein synthesis machinery.
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            Dynamic nuclear pore complexes: life on the edge.

            The exchange of molecules between the nucleus and cytoplasm is mediated through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Altering the interactions between transport receptors and their cargo has been shown to be a major regulatory mechanism to control traffic through NPCs. New evidence now suggests that NPC proteins play active roles in translocation, and that transport is also controlled by dynamic changes in NPC composition and architecture. This view of ever-changing NPCs necessitates the re-evaluation of current models of nuclear transport and how this process is regulated.
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              NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1.

              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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                Author and article information

                Journal
                J Biol Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                19 August 2011
                27 June 2011
                27 June 2011
                : 286
                : 33
                : 29325-29335
                Affiliations
                [1]From the Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom
                Author notes
                [2 ] To whom correspondence should be addressed: MRC-LMB, Hills Rd., Cambridge, CB2 0QH, UK Fax: 44-1223-213556; E-mail: ms@ 123456mrc-lmb.cam.ac.uk .
                [1]

                Supported by a research studentship from the Woolf Fisher Trust of New Zealand.

                Article
                M111.245092
                10.1074/jbc.M111.245092
                3190738
                21708948
                d3fe2066-4f59-4d06-b867-1ef03226674f
                © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 30 March 2011
                : 17 June 2011
                Categories
                Cell Biology

                Biochemistry
                x-ray scattering,protein structure,nuclear pore,protein-protein interactions,protein conformation,nuclear transport,trafficking

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