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• W2002245757 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. 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. Nuclear protein export is a fundamental function in all eukaryotes and is crucial for maintaining the appropriate nucleocytoplasmic distribution of a broad range of transcription factors, signaling molecules, and cell cycle regulators, as well as viral components such as the HIV Rev protein and the influenza NS2 protein. Transport takes place through nuclear pore complexes and is mediated primarily by the transport factor CRM1 (Xpo1p in yeast) in conjunction with the Ran GTPase (1Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1739) Google Scholar, 2Kutay U. Izaurralde E. Bischoff F.R. Mattaj I.W. Görlich D. EMBO J. 1997; 16: 1153-1163Crossref PubMed Scopus (311) Google Scholar, 3Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 4Weis K. Cell. 2003; 112: 441-451Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 5Kutay U. Güttinger S. Trends Cell Biol. 2005; 15: 121-124Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 6Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 7Strambio-De-Castillia C. Niepel M. Rout M.P. Nat. Rev. Mol. Cell Biol. 2010; 11: 490-501Crossref PubMed Scopus (370) Google Scholar). Proteins destined for export have nuclear export signal sequences (NESs) 3The abbreviations used are: NESnuclear export signalSAXSsmall angle x-ray scatteringNi-NTAnickel-nitrilotriacetic acidTEVtobacco etch mosaic virus. and bind cooperatively to CRM1 with RanGTP in the nucleus. This cargo-carrier complex is then equilibrated between the nucleus and cytoplasm through weak interactions between CRM1 and nuclear pore proteins that have characteristic repeating phenylalanine-glycine sequence motifs (FG-nucleoporins). When the cargo-carrier complex reaches the cytoplasm, RanGTP is removed through the action of RanBP1, RanGAP, and several nucleoporins. When RanGTP is removed, the affinity of CRM1 for the NES is reduced, leading to the release of the cargo, after which CRM1 is recycled to the nucleus (reviewed by 4–8). Energy for the cycle is ultimately provided by GTP hydrolysis on Ran that is stimulated by RanGAP. nuclear export signal small angle x-ray scattering nickel-nitrilotriacetic acid tobacco etch mosaic virus. CRM1 is a member of the β-karyopherin superfamily and is constructed from a tandem array of 21 HEAT repeats, each of which is based on two α-helices joined by loops of varying length (8Cook A.G. Conti E. Curr. Opin. Struct. Biol. 2010; 20: 247-252Crossref PubMed Scopus (59) Google Scholar, 9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar, 10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar, 11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar, 12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar). The molecule adopts an approximately toroidal conformation with one series of HEAT helices (the A-helix) located on the outer convex surface of the toroid, whereas the other (the B-helix) is located on its inner concave surface (8Cook A.G. Conti E. Curr. Opin. Struct. Biol. 2010; 20: 247-252Crossref PubMed Scopus (59) Google Scholar, 9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar, 10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar, 11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar, 12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar). The α-helix at the CRM1 C terminus (referred to as HEAT repeat 21B by Monecke et al. (10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar)) adopts different positions depending on whether or not RanGTP is bound. In the absence of RanGTP (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar), the C-terminal helix projects across the central cavity of the CRM1 toroid, whereas when RanGTP is bound (10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar, 11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar, 12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar), the helix is displaced so that its tip sits near HEAT repeats 3 and 4 in the N-terminal region of the CRM1 toroid. Complexes of CRM1 with Snurportin (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar, 10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar) and chimeras of Snurportin with a range of NESs (12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar) have shown that the NES-binding site is located on the outer surface of CRM1 in a hydrophobic groove formed between the A-helices of HEAT repeats 11 and 12. RanGTP, on the other hand, binds to the interior surface of the CRM1 toroid, making connections with the N-terminal half of CRM1, the central acidic loop HEAT repeat 9, and HEAT repeats 17 and 19 (10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar, 11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar, 12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar). Detailed structural studies of CRM1 with a range of different NESs bound has established a consensus for NESs and accounted for how CRM1 is able to recognize the range of different NESs observed (12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar). Because the binding and release of NESs in the nucleus and cytoplasm, respectively, is critical for nuclear protein export, it is important to understand the molecular mechanism by which the affinity of CRM1 for NESs is modulated. In the nucleus when RanGTP is bound, CRM1 has a higher affinity for NESs than in the cytoplasm when Ran has been removed. This change in affinity could be due to changes in the positions of the A-helices of HEAT repeats 11 and 12 that result from a contribution of local changes induced via an autoinhibitory mechanism of the HEAT 9 loop proposed by Koyama and Matsuura (11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar) and/or a larger, global change in CRM1 conformation as proposed by Dong et al. (13Dong X. Biswas A. Chook Y.M. Nat. Struct. Mol. Biol. 2009; 16: 558-560Crossref PubMed Scopus (76) Google Scholar) and Monecke et al. (10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar). In addition to the role of the HEAT 9 loop in modulating the affinity of CRM1 for NESs, the C-terminal helix also influences NES binding. Although in the absence of RanGTP full-length CRM1 has weak affinity for NESs, deletion of the C-terminal helix increases this affinity to a level that, in pulldown binding studies, appeared comparable with that seen in the presence of RanGTP (13Dong X. Biswas A. Chook Y.M. Nat. Struct. Mol. Biol. 2009; 16: 558-560Crossref PubMed Scopus (76) Google Scholar). Here we use a combination of mutagenesis and small angle x-ray scattering (SAXS) to investigate the role of the C-terminal helix in greater detail and show that charged residues at its extreme C terminus are critical to its influence on the affinity of CRM1 for NESs. Deleting the last 9 residues of mouse CRM1 or mutating to alanine either the acidic residues in this region or a cluster of positive residues in the B-helix of HEAT repeat 12 (Lys-594, Arg-596, and Arg-597) increased the affinity of CRM1 for the PKI NES. Moreover, the behavior of a variant in which the charges of both clusters were reversed reverted to the affinity seen with wild type CRM1. SAXS indicated that the C-terminal helix was positioned across the CRM1 toroid and that none of the engineered variants generated a substantial conformational change in the molecule. Based on these results, we propose a model in which the C-terminal helix and the HEAT loop 9 induce local conformational changes that stabilize the closed conformation of the CRM1 NES binding site, which contributes to the release of NES-containing cargoes into the cytoplasm following the removal of Ran and to preventing the return of cargoes to the nucleus. S. cerevisiae Xpo1p was expressed from the pET30TB plasmid (a derivative of pET30a-TEV in which the BamHI site in the TEV sequence has been removed) that adds a His6-S tag followed by a TEV cleavage site to the N terminus (14Matsuura Y. Stewart M. Nature. 2004; 432: 872-877Crossref PubMed Scopus (161) Google Scholar). Mouse CRM1 was expressed as a TEV-cleavable His6-ZZ-tagged construct in a derivative of a pQE80 vector that was obtained as a kind gift from Dr. Dirk Görlich (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany). Mutations in CRM1/Xpo1p were introduced via overlapping PCRs with mutagenic primers using the Herculase II fusion system (Stratagene). Proteins were expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells in ZYM-5052 autoinduction medium (15Studier F.W. Protein Expr. Purif. 2005; 41: 207-234Crossref PubMed Scopus (4109) Google Scholar). A single colony of BL21-CodonPlus(DE3)-RIL cells that had been transformed with the desired plasmid was used to inoculate a starter culture of 10 ml of LB medium (that contained either 100 ng/ml ampicillin or 25 ng/ml kanamycin as appropriate). The culture was incubated overnight at 37 °C, shaking at 200 rpm, and was used to inoculate a large-scale 1-liter culture the following morning. Large scale cultures were grown in 2-liter conical flasks that contained ∼1 liter of ZYM-5052 medium. To prepare the final media, a stock of ∼1 liter of ZY medium (10 g of N-Z-amine AS and 5 g of yeast extract dissolved in 1 liter of MilliQ water) was supplemented with 20 ml of 50× 5052 solution (25% (w/v) glycerol, 2.5% (w/v) glucose, and 10% (w/v) α-lactose monohydrate), 20 ml of 50× M solution (1.25 m Na2HPO4, 1.25 m KH2PO4, 2.5 m NH4Cl, and 0.25 m Na2SO4), 2 ml of 1 m MgSO4, and 100 mg of either ampicillin or kanamycin as appropriate. Stocks of the 50× 5052 and 50× M solutions were sterile filtered and stored at 4 °C. The cultures were incubated at 37 °C, shaking at 200 rpm, for ∼6 h that allowed growth to an A600 ∼ 0.5, at which point the temperature was lowered to 20 °C for a further 18 h. The cells were harvested by centrifugation at ∼4,000 × g for 10 min at 4 °C, and cell pellets were stored at −20 °C. Frozen cell pellets were thawed at room temperature and resuspended in 10 ml of the appropriate lysis buffer per cell pellet from 1 liter of culture. A CompleteTM Mini EDTA-free protease inhibitor mixture tablet (Roche Applied Science) was added and dissolved in the lysis mixture. Cell lysis was carried out by high pressure cavitation using an EmulsiFlex C5 homogenizer (Avestin). Lysates were clarified by centrifugation at 45,000 × g for 20 min at 4 °C. Proteins were purified using a standard Ni-NTA resin (Qiagen) affinity purification protocol. Buffers typically contained 15 mm imidazole, pH 8.0, to prevent nonspecific binding of contaminant proteins. Clarified lysate containing soluble His6-tagged protein was mixed with the Ni-NTA resin and incubated at room temperature for 45 min. The resin was centrifuged at 1,500 rpm for 5 min at 4 °C and washed three times with 50 ml of binding buffer. Purified protein was eluted from the resin using binding buffer that contained at least 300 mm imidazole, pH 8.0. All of the protein constructs used in this work contained a tobacco etch mosaic virus (TEV) protease cleavage site. TEV cleavage was typically conducted overnight at 4 °C using a His6-tagged S219V variant of TEV that is less prone to autocatalysis and is more active at cold temperatures than wild type TEV protease (16Kapust R.B. Tözsér J. Fox J.D. Anderson D.E. Cherry S. Copeland T.D. Waugh D.S. Protein Eng. 2001; 14: 993-1000Crossref PubMed Scopus (632) Google Scholar). Following TEV cleavage, His6 tags and the His6-TEV S129V were removed from cleavage mixtures by a second incubation with fresh Ni-NTA resin. Gel filtration chromatography was carried out as a final protein purification step to remove contaminating proteins and CRM1 oligomers using a HiLoad Superdex 200 26/60 prep grade column (GE Healthcare). Protein fractions were analyzed using SDS-PAGE, and those that contained the target protein of sufficient purity (>95%) were pooled and concentrated using an Amicon-Ultra 15 centrifugal filter unit (Millipore) following the manufacturer's instructions. Proteins were concentrated to at least 50 mg/ml and stored at either 4 °C for immediate use or flash-frozen in liquid nitrogen and stored for longer term use at −80 °C. His-tagged-YFP-PKI-NES was prepared as described (11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar). Pulldown assays were used to assess the binding between proteins by immobilizing one protein on affinity resin via its affinity purification tag. A small aliquot (∼100 μl) of affinity resin (Ni-NTA or glutathione-Sepharose 4B (GE Healthcare)) was washed with binding buffer (typically 20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 2 mm DTT). The protein that would act as bait in the binding assay was then loaded onto the beads. Either clarified lysate (∼0.5 ml) or purified protein was added to the resin and incubated at room temperature for 45 min. For GST pulldowns, a negative control was conducted in parallel by performing the assay on resin loaded with GST protein alone. For Ni-NTA pulldowns, resin without bait protein was used as a negative control for nonspecific binding to the beads. After incubation, beads were sedimented by centrifugation at 2,000 rpm for 3 min in a microcentrifuge and washed three times with 1 ml of binding buffer to remove excess bait protein. The prey proteins were added to the resin (in quantities that provided a molar excess to the bait protein), and the mixture was incubated for 45 min at room temperature. Unbound fractions were removed, and the resin was washed three times with 0.5 ml of binding buffer. After the final wash step, the beads were mixed with an equal volume of 2× SDS-PAGE loading dye and boiled at 95 °C to release bound proteins from the beads. Protein samples were analyzed by SDS-PAGE. The binding of increasing concentrations of wild type and mutant CRM1 protein to 10–50 nm YFP-PKI-NES fusion protein (11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar) was measured by fluorescence anisotropy at 25 °C using a PerkinElmer LS55 fluorimeter equipped with a Hamilton Microlab titrator controlled by laboratory software. The reaction was followed in 20 mm Tris-HCl, pH 7.4, 50 mm NaCl, 5 mm DTT, 2 mm MgCl2. Excitation and emission wavelengths of 507 and 526 nm, respectively, were used with both excitation and emission slits set at 5 nm. Using a single-site binding model, the data were fitted to the equation, F=F0+(F1-F0){([PT]+[LT]+Kd)-([PT]+[LT]+KD)2-4[PT][LT]}2[PT](Eq. 1) where F0 and F1 are the anisotropy in the absence of titrating protein and at saturation, respectively; [LT] and [PT] are the total concentrations of protein and YFP-NES, respectively; and Kd is the dissociation constant. The fraction of YFP-NES bound at each concentration of titratant, fb, was calculated using the fitted constants from the equation above using the following. fb=F-F0F1-F0(Eq. 2) The CRM1 variants gave excellent binding isotherms that were fitted well by the model (supplemental Fig. S1). However, low (∼15 μm) affinity of wild type CRM1 for the YFP-NES precluded our being able to attain saturation, which was taken to be the same as that observed for the variants. SAXS data were collected at European Synchrotron Radiation Facility Beamline ID14-3 using a fixed wavelength of 0.931 Å. CRM1 or Xpo1p samples were diluted with the original gel filtration buffers to make a series of protein concentrations. The signal from protein scattering was collected in conjunction with a buffer sample measurement taken before and after each protein sample. For mouse CRM1, the detector was a Pilatus 1 m (Dectris), and the sample to the detector distance was 2.425 m. The momentum transfer range measured was 0.05 < s < 6 nm−1. The samples were exposed for 10 frames of 10 s each. For Saccharomyces cerevisiae Xpo1p, the detector was a VÅNTEC-2000 (Bruker AXS Ltd.), and the sample to the detector distance was 1.668 m. The momentum transfer range measured was 0.05 < s < 6 nm−1. The samples were exposed for 10 frames of 30 s each. SAXS data were analyzed using the PRIMUS software suite (17Konarev P.V. Volkov V.V. Sokolova A.V. Koch M.H. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 1277-1282Crossref Scopus (2342) Google Scholar). Each protein sample frame was inspected for radiation damage, and frames that contained significant damage were omitted. The buffer data were averaged from before and after the protein sample and subtracted from the merged protein dataset to provide a final curve for that protein concentration. The radius of gyration, Rg, was calculated from the slope of the Guinier region with output from the AUTORG program used as a guide to the best data range for this measurement. The pair distribution function, p(r), was calculated using the program GNOM that performs an indirect Fourier transform. An initial estimate of the Dmax was provided to GNOM, and the program was run several times to determine the best Dmax based on the shape of the p(r) function. The program CRYSOL was used to provide a direct comparison between the calculated scattering profile from a given Protein Data Bank coordinate file and an experimental SAXS profile (18Svergun D.I. Barberato C. Koch M.H. J. Appl. Crystallogr. 1995; 28: 768-773Crossref Scopus (2766) Google Scholar). CRYSOL calculates the scattering, taking into account the hydration shell of the particle. It has been shown that no two unique protein structures have identical calculated scattering profiles (19Sokolova A.V. Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 865-868Crossref Scopus (28) Google Scholar). The final output provides a χ2 value to give a measure of the goodness of fit between the calculated and experimental scattering curves. Deletion of the C-terminal 44 residues of human CRM1 (that correspond to the C-terminal helix) has been shown to increase its affinity for NESs in the absence of RanGTP, which, in pulldown binding studies, appeared to be of a level comparable with that of wild type CRM1 in the presence of RanGTP (13Dong X. Biswas A. Chook Y.M. Nat. Struct. Mol. Biol. 2009; 16: 558-560Crossref PubMed Scopus (76) Google Scholar). Sequence alignment of CRM1 from several species shows a strongly conserved pattern within the residues at the distal tip of the C-terminal helix, whereby a single basic residue is followed by a series of acidic residues (Fig. 1). In CRM1 from Homo sapiens, Mus musculus, and Xenopus laevis, there are four acidic residues, but the precise number can vary between three (in S. cerevisiae) and six (in Caenorhabditis elegans). Because there is no electron density corresponding to these acidic residues in any of the current crystal structures of CRM1 or Xpo1p, these residues are not present in any of the structural models (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar, 10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar, 11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar, 12Güttler T. Madl T. Neumann P. Deichsel D. Corsini L. Monecke T. Ficner R. Sattler M. Görlich D. Nat. Struct. Mol. Biol. 2010; 17: 1367-1376Crossref PubMed Scopus (180) Google Scholar). In the crystal structure of the human CRM1-Snurportin complex (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar), the C-terminal helix lies across the central cavity of the toroid, bringing the distal tip residues of the helix near to HEAT repeats 11 and 12. These HEAT repeats also form the primary binding site for NESs in a groove formed between their A-helices. The role of the acidic residues at the tip of the CRM1 C-terminal helix in modulating the affinity of CRM1 for NESs was probed using a series of deletion variants, which were evaluated for their affinity for immobilized GST-PKI NES (Fig. 2). Because it could be expressed to higher levels and appeared not to aggregate so extensively in solution, we employed mouse CRM1 rather than the human CRM1 employed previously (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar). The sequences of these proteins are identical in the final 10 residues of the C-terminal helix (Fig. 1). CRM1 variants were generated in which the entire C-terminal helix was deleted (CRM1-1–1027; as in Dong et al. (13Dong X. Biswas A. Chook Y.M. Nat. Struct. Mol. Biol. 2009; 16: 558-560Crossref PubMed Scopus (76) Google Scholar)); the distal half was deleted (CRM1-1–1040) so that it retained the base of the C-terminal helix, which in the CRM1-Snurportin-RanGTP (10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar) ternary complex makes contacts with residues in the inter-repeat loops of N-terminal HEAT repeats 3 and 4, or in which the last nine residues, that include the conserved acidic residues, were deleted (CRM1-1–1062). A CRM1 variant (CRM1-tipA) in which the charged residues of the final nine residues of CRM1 were replaced with alanines was also tested. All four of these mouse CRM1 variants bound the PKI NES with a higher affinity than wild type CRM1 (Fig. 2). Determination of binding constants using fluorescence anisotropy (Table 1) indicated that although deleting the entire C-terminal helix produced a larger increase in affinity for the PKI NES, simply removing the negative charge from the tip of the C-terminal helix caused over a 10-fold increase in affinity, consistent with the charged residues at the tip making a substantial contribution to the influence of the C-terminal helix on the affinity of CRM1 for NESs.TABLE 1Affinity of CRM1 and variants for YFP-PKI-NES determined using fluorescence anisotropyCRM1 proteinKdΔΔGμmkcal/molWild-type15.6 ± 2.31–10621.7 ± 0.11.3 ± 0.2VLV > AAA0.20 ± 0.032.6 ± 0.21–10270.30 ± 0.052.3 ± 0.21–1062 + VLV > AAA0.025 ± 0.0023.8 ± 0.2 Open table in a new tab The important contribution to modulation of the affinity of CRM1 for the PKI NES made by the highly conserved patch of acidic residues at the tip of the C-terminal helix suggested that complementary positively charged residues on CRM1 might mediate an electrostatic interaction with this cluster. Examination of the electrostatic potential of the inner surface of CRM1 in the structure of the human CRM1-Snurportin complex (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar), showed a basic patch located directly adjacent to where the C-terminal helix terminates (Fig. 3A). This patch is formed by a series of conserved basic residues, which sit at the ends of the B-helices of HEAT repeats 11 and 12 that are closest to the end of the C-terminal helix (Figs. 3B and 4). These conserved residues are Arg-553 and Arg-556 of HEAT 11B and Lys-594, Arg-596, and Arg-597 of HEAT 12B. Lys-590 is also conserved and has been shown to bind to residue Glu-429 from the HEAT 9 loop in the Xpo1p-RanGTP-RanBP1 crystal structure (11Koyama M. Matsuura Y. EMBO J. 2010; 29: 2002-2013Crossref PubMed Scopus (78) Google Scholar). In the mouse CRM1-Snurportin-RanGTP structure (10Monecke T. Güttler T. Neumann P. Dickmanns A. Görlich D. Ficner R. Science. 2009; 324: 1087-1091Crossref PubMed Scopus (170) Google Scholar), Lys-590 binds to Glu-428 of the HEAT 9 loop rather than Glu-429, which instead binds to Tyr-155 of RanGTP. The proximity of the acidic C-terminal helix tip to the conserved basic patch located on the HEAT 11/12 B helices was consistent with an electrostatic interaction between these two sets of residues having the potential to contribute to the influence of the C-terminal helix of CRM1 on its affinity for NESs in the absence of RanGTP.FIGURE 4Conserved basic residues on HEAT 11B/12B of CRM1. The conserved basic residues (Arg-553, Arg-556, Lys-590, Lys-594, Arg-596, and Arg-597) shown in dark blue lie at the end of the HEAT 11B/12B helices closest to the end of the C-terminal helix (as modeled by Dong et al. (9Dong X. Biswas A. Süel K.E. Jackson L.K. Martinez R. Gu H. Chook Y.M. Nature. 2009; 458: 1136-1141Crossref PubMed Scopus (252) Google Scholar)). The NES binds on the opposite side of the CRM1 backbone at the A helices of these HEAT repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We tested this hypothe" @default.
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