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• W1995160911 abstract "Importin-α is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-α, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-α and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX 10–12KRRK) that can be used to identify novel nuclear proteins. Importin-α is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-α, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-α and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX 10–12KRRK) that can be used to identify novel nuclear proteins. Nucleocytoplasmic transport occurs through nuclear pore complexes, large proteinaceous structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (∼25 MDa). The best characterized nuclear targeting signals are the conventional nuclear localization sequences (NLSs) 1The abbreviations used are: NLS, nuclear localization sequence; Arm repeat, armadillo repeat; Impα, importin-α; Impβ, importin-β; m-Impα, mouse importin-α (residues 70–529); y-Impα, yeast importin-α (residues 88–530); N1N2, X. laevis phosphoprotein N1N2; RB, human retinoblastoma protein; T-Ag, simian virus 40 large T-antigen.1The abbreviations used are: NLS, nuclear localization sequence; Arm repeat, armadillo repeat; Impα, importin-α; Impβ, importin-β; m-Impα, mouse importin-α (residues 70–529); y-Impα, yeast importin-α (residues 88–530); N1N2, X. laevis phosphoprotein N1N2; RB, human retinoblastoma protein; T-Ag, simian virus 40 large T-antigen. that contain one or more clusters of basic amino acids (1Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1710) Google Scholar). The NLSs fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters separated by a spacer. Despite the variability, the conventional basic NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of α and β subunits (for recent reviews, see Refs. 2Conti E. Results Probl. Cell Differ. 2002; 35: 93-113Crossref PubMed Scopus (18) Google Scholar, 3Damelin M. Silver P.A. Corbett A.H. Methods Enzymol. 2002; 351: 587-607Crossref PubMed Scopus (35) Google Scholar, 4Weis K. Curr. Opin. Cell Biol. 2002; 14: 328-335Crossref PubMed Scopus (165) Google Scholar). Importin-α (Impα) contains the NLS-binding site, and importin-β (Impβ) is responsible for the translocation of the importin-substrate complex through the nuclear pore complex. Once inside the nucleus, Ran-GTP binds to Impβ and causes the dissociation of the import complex. Impα becomes autoinhibited, and both importin subunits return to the cytoplasm separately without the import cargo. The directionality of nuclear import is conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins. Impα consists of two structural and functional domains, a short basic N-terminal Impβ-binding domain (5Gorlich D. Henklein P. Laskey R.A. Hartmann E. EMBO J. 1996; 15: 1810-1817Crossref PubMed Scopus (361) Google Scholar, 6Weis K. Ryder U. Lamond A.I. EMBO J. 1996; 15: 1818-1825Crossref PubMed Scopus (223) Google Scholar, 7Moroianu J. Blobel G. Radu A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6572-6576Crossref PubMed Scopus (84) Google Scholar) and a large NLS-binding domain built of armadillo (Arm) repeats (8Peifer M. Berg S. Reynolds A.B. Cell. 1996; 76: 789-791Abstract Full Text PDF Scopus (546) Google Scholar). The structural basis of monopartite and bipartite NLS recognition by Impα has been studied crystallographically in yeast and mouse Impα proteins (9Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 10Conti E. Kuriyan J. Structure. 2000; 8: 329-338Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar). The two basic clusters of the bipartite NLSs bind to two separate binding sites on Impα, involving Arm repeats 1–4 and 4–8, respectively. Monopartite NLSs can bind in both sites but primarily use the binding site corresponding to the C-terminal basic cluster of the bipartite NLSs, referred to as the major site (9Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar). The structure of full-length Impα indicated that the major NLS-binding site is occupied by residues 44–54 from the N-terminal region of the protein (Impβ-binding domain) that resembles an NLS (12Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (319) Google Scholar); Impα is therefore autoinhibited in the absence of Impβ. This observation is supported by the measurements of higher NLS binding affinity by Impα/β as compared with Impα alone (13Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 14Gorlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (528) Google Scholar, 15Efthymiadis A. Shao H. Hübner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Hübner S. Xiao C.Y. Jans D.A. J. Biol. Chem. 1997; 272: 17191-17195Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 17Hübner S. Smith H.M.S. Hu W. Chen C.K. Rihs H.P. Paschal B.M. Raikhel N.V. Jans D.A. J. Biol. Chem. 1999; 274: 22610-22617Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Briggs L.J. Stein D. Goltz J. Corrigan V.C. Efthymiadis A. Hübner S. Jans D.A. J. Biol. Chem. 1998; 273: 22745-22752Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 19Hu W. Jans D.A. J. Biol. Chem. 1999; 274: 15820-15827Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Fanara P. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2000; 275: 21218-21223Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 21Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2001; 276: 1317-1325Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 22Catimel B. Teh T. Fontes M.R. Jennings I.G. Jans D.A. Howlett G.J. Nice E.C. Kobe B. J. Biol. Chem. 2001; 276: 34189-34198Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The significance of the autoinhibitory mechanism has been confirmed by in vivo studies (23Harreman M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In this study, we present the crystal structures of peptides corresponding to the bipartite NLSs from human retinoblastoma protein (RB) and Xenopus laevis chromatin assembly factor N1N2 bound to mouse Impα. These NLSs were chosen to represent diverse sequences and different lengths of the linkers between the clusters, so that some general conclusions can be drawn on NLS binding. The basic clusters of both peptides bind in the expected binding pockets, but the linker regions make specific contacts with the receptor also. Comparisons with other available Impα structures allow us to explain the specificities of monopartite and bipartite NLS binding and help us improve the definition of the consensus sequence of a conventional basic/bipartite NLS. The results will have general implications for recognizing the NLSs in new gene products identified in genome sequences and therefore for functional annotation of new proteins. Peptide Synthesis—The peptides CGKRSAEGSNPPKPLKKLRGY (RB peptide) and CGRKKRKTEEESPLKDKAKKSKGY (N1N2 peptide) were synthesized using the Applied Biosystems 433A peptide synthesizer, purified by cation exchange chromatography followed by reverse phase chromatography, and analyzed by quantitative amino acid analysis using a Beckman 6300 amino acid analyzer and electrospray mass spectrometry (Sciex API 111, PerkinElmer Life Sciences) (24Michell B.J. Stapleton D. Mitchelhill K.I. House C.M. Katsis F. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 28445-28450Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The peptides RB and N1N2 correspond to the NLSs of human retinoblastoma protein, residues 861–877, and X. laevis N1N2 protein (N1N2), residues 535–555, respectively, with two heterologous residues added at each terminus. Protein Expression, Purification, and Crystallization—N-terminal truncated mouse importin α (α2 isoform (25Kussel P. Frasch M. Mol. Gen. Genet. 1995; 248: 351-363Crossref PubMed Scopus (51) Google Scholar)) lacking 69 N-terminal residues (m-Impα) was expressed recombinantly in Escherichia coli as a fusion protein containing a hexa-histidine tag (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar). For crystallization, m-Impα was concentrated to 18.8 mg/ml (in 20 mm Tris-HCl (pH 8.0), 100 mm NaCl, and 10 mm dithiothreitol) using a Centricon-30 (Millipore) and stored at –20 °C. Crystallization conditions were screened by systematically altering various parameters using the crystallization conditions successful for other peptide complexes (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar) as a starting point. The crystals of both complexes (rod-shaped, 0.5 × 0.2 × 0.1 mm for the RB peptide complex and 0.4 × 0.1 × 0.07 mm for the N1N2 peptide complex) were obtained using co-crystallization by combining 1 μl of protein solution, 0.7 μl of peptide solution (1.7 mg/ml with peptide/protein ratio 3.5), and 1 μl of reservoir solution and suspended over 0.5 ml of reservoir solution containing 0.6 m sodium citrate (pH 6.0) and 10 mm dithiothreitol. Diffraction Data Collection—The crystals exhibit orthorhombic symmetry (space group P212121; Table I). Diffraction data were collected from single crystals transiently soaked in a solution analogous to the reservoir solution but supplemented with 23% glycerol and flash-cooled at 100 K in a nitrogen stream (Oxford Cryosystems), using a MAR-Research image plate detector (plate diameter, 345 mm) and CuKα radiation from a Rigaku RU-200 rotating anode generator. Data were autoindexed and processed with the HKL suite (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38446) Google Scholar) (Table I).Table IStructure determinationRB peptideN1N2 peptideDiffraction data statisticsUnit cell dimensions (Å)a79.078.9b89.289.7c101.0101.0Resolution (Å)99-2.5 (2.59-2.5)aNumbers in parentheses are for the highest resolution shell.99-2.5 (2.59-2.5)aNumbers in parentheses are for the highest resolution shell.Observations249,870211,968Unique reflections25,38225,538Completeness (%)96.0 (98.3)95.5 (94.6)R mergebR merge = Σhkl(Σi(Ihkl,i - <Ihkl >))/Σhkl,i <Ihkl >, where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k and l, and <Ihkl > is the mean intensity of that reflection. Calculated for I > - 3σ(I). (%)7.3 (40.3)8.2 (51.5)Average I/σ(I)19.0 (2.8)15.7 (1.8)Refinement statisticsResolution (Å)30-2.5 (2.66-2.5)30-2.5 (2.66-2.5)Number of reflections (F>0)24,323 (4,075)24,340 (4,210)Completeness (%)96.0 (98.3)95.5 (94.5)R crystcR cryst = Σhkl(∥F(obs)hkl - F(calc)hkl∥)/ F(obs)hkl, where F(obs)hkl and F(calc)hkl are the observed and calculated structure factor amplitudes, respectively. (%)20.4 (26.3)20.9 (30.2)R freedR free is equivalent to R cryst but calculated with reflections (5%) omitted from the refinement process. (%)23.9 (29.2)23.3 (32.6)Number of non-hydrogen atomsProtein3,2613,244Peptide146159Water173123Mean B-factor (Å2)46.247.2r.m.s deviations from ideal valueseCalculated with the program CNS (27).Bond lengths (Å)0.0060.007Bond angles (o)1.21.4Ramachandran plotfCalculated with the program PROCHECK (30).Residues in most favored (disallowed) regions (%)93.8 (0.3)91.9 (0.3)Coordinate error (Å)eCalculated with the program CNS (27).Luzzati plot (cross-validated Luzzati plot)0.28 (0.35)0.29 (0.37)SIGMAA (cross-validated SIGMAA)0.24 (0.29)0.25 (0.29)a Numbers in parentheses are for the highest resolution shell.b R merge = Σhkl(Σi(Ihkl,i - <Ihkl >))/Σhkl,i <Ihkl >, where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k and l, and <Ihkl > is the mean intensity of that reflection. Calculated for I > - 3σ(I).c R cryst = Σhkl(∥F(obs)hkl - F(calc)hkl∥)/ F(obs)hkl, where F(obs)hkl and F(calc)hkl are the observed and calculated structure factor amplitudes, respectively.d R free is equivalent to R cryst but calculated with reflections (5%) omitted from the refinement process.e Calculated with the program CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar).f Calculated with the program PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Structure Determination and Refinement—The crystals of the peptide complexes were highly isomorphous with the crystals of full-length Impα (12Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (319) Google Scholar); therefore, the structure of mouse Impα (Protein Data Bank number 1IAL) with N-terminal residues omitted was used as a starting model for crystallographic refinement. Electron density maps were inspected for the presence of the peptide after rigid body refinement using the program CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar) (RB peptide: m-Impα complex, R cryst = 30.5%, R free = 32.4%, 6–4-Å resolution; N1N2 peptide: m-Impα complex, R cryst = 30.8%, R free = 35.1%, 6–4-Å resolution; Table I provides the explanation of R-factors). Electron density maps calculated with coefficients 3 F obs–2 F calc and simulated annealing omit maps (Fig. 1) calculated with analogous coefficients were generally used. The model was improved, as judged by the free R-factor (28Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3855) Google Scholar), through rounds of crystallographic refinement (positional and restrained isotropic individual B-factor refinement with an overall anisotropic temperature factor and bulk solvent correction) and manual rebuilding (program O (29Jones T.A. Bergdoll M. Kjeldgaard M. Bugg C.E. Ealick S.E. Crystallographic and Modeling Methods in Molecular Design. Springer-Verlag, New York1990: 189-195Crossref Google Scholar)). Solvent molecules were added with the program CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar). Asn239 is an outlier in the Ramachandran plot as also observed in all other structures of mouse Impα (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar, 12Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (319) Google Scholar). Pro242 is a cis-proline. The final models comprise 427 Impα residues (residues 71–497), 20 peptide residues, and 173 water molecules for RB peptide-Impα complex, and 426 Impα residues (residues 72–496), 21 peptide residues, and 123 water molecules for N1N2 peptide-Impα complex (Table I). The coordinates have been deposited in the Protein Data Bank (Protein Data Bank numbers 1PJM and 1PJN for the RB and N1N2 peptide complexes, respectively). Structure Analysis—The quality of the models was assessed with the program PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). The contacts were analyzed with the program CONTACT, and the buried surface areas were calculated using the program CNS (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar). Bioinformatic Analysis—We used the consensus sequence KRRK to search for NLS-containing proteins using the Quick Matrix option of Scansite (31Yaffe M.B. Leparc G.G. Lai J. Obata T. Volinia S. Cantley L.C. Nat. Biotechnol. 2001; 19: 348-353Crossref PubMed Scopus (463) Google Scholar); the sequence KRXK was entered for the primary preference positions 0 to +3, and R was entered into the secondary preference position +2. The bipartite consensus was too long to use with the current version of Scansite. Testing using proteins with known NLSs showed that there is a high likelihood of detecting a functional NLS at Scansite scores ≤ 0.0408 (corresponding to 0.448% of all yeast proteins) and a reasonable likelihood at Scansite scores ≤ 0.0596 (corresponding to 1.169% of all yeast proteins). To estimate the efficiency of detecting an unknown NLS, we performed a Scansite search with a test set of 50 randomly selected yeast nuclear proteins (Munich Information Center for Protein Sequences subcellular catalogue (32Mewes H.W. Frishman D. Guldener U. Mannhaupt G. Mayer K. Mokrejs M. Morgenstern B. Munsterkotter M. Rudd S. Weil B. Nucleic Acids Res. 2002; 30: 31-34Crossref PubMed Scopus (759) Google Scholar)); 9 proteins showed a sequence match to the above motif with Scansite scores ≤ 0.0408, and 23 proteins (46%) showed a match with Scansite scores ≤ 0.0569. For comparison, we searched for NLSs in the same test set of proteins using PredictNLS (33Cokol M. Nair R. Rost B. EMBO Rep. 2000; 1: 411-415Crossref PubMed Scopus (550) Google Scholar); this method detected an NLS in nine proteins, some of which did not belong to the conventional basic/bipartite group. Structure Determination—The RB and N1N2 NLS peptides were co-crystallized with an N-terminal truncated mouse Impα lacking residues 1–69 (m-Impα); residues 1–69 are responsible for autoinhibition. The co-crystals with both peptides grew in similar conditions and isomorphously to other mouse Impα crystals (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar, 12Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (319) Google Scholar). Electron density maps based on the Impα model, following rigid body refinement, clearly showed electron density corresponding to the peptides (Fig. 1). The structures were refined at 2.5-Å resolution for both complexes (Table I). Residues 859–878 of the peptide RB (residue 879 had no interpretable electron density) and residues 535–555 of the peptide N1N2 (residues 533–534 and 556 had no interpretable electron density) could unambiguously be identified in the electron density maps (Fig. 1). The side chains of Glu543 and Lys549 of N1N2 were poorly ordered; therefore, these residues were modeled as alanines. Structure of Importin-α in the Complexes—Impα forms a single elongated domain built from 10 Arm structural repeats, each containing three α helices (H1, H2, and H3) connected by loops (Fig. 2). The structure of Impα in the complexes is comparable with the crystal structure of the full-length Impα. (r.m.s. deviations of Cα atoms of Impα residues 72–496 are 0.22 Å between the RB and N1N2 peptide-m-Impα complexes; 0.35 and 0.31 Å between full-length Impα and the RB and N1N2 peptide-m-Impα complexes, respectively; and 0.34 and 0.30 Å between nucleoplasmin peptide-m-Impα and the RB and N1N2 peptide-m-Impα complexes, respectively.) Binding of the NLS Peptides to Importin-α—The peptides bind in an extended conformation with the chain running antiparallel to the direction of the Arm repeat superhelix (Fig. 2). The base of the groove that contains the binding sites is formed mainly by the H3 helices of the Arm repeats, which carry some residues conserved among the repeats, including the tryptophans and asparagines at the third and fourth turns in H3 helices of the Arm repeats, respectively (10Conti E. Kuriyan J. Structure. 2000; 8: 329-338Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar). The two basic clusters of the RB and N1N2 peptides bind to two separate well defined binding sites on the surface of the m-Impα molecule, referred to as the minor and major sites (Fig. 2). The minor site specifically binds to the N-terminal basic cluster KR, and the larger, C-terminal basic cluster binds to the major site. The electron density is present for 20 peptide residues in the RB peptide (average B-factor, 63.3 Å2) and for 21 peptide residues of the N1N2 peptide (average B-factor, 72.1 Å2) (Fig. 1). In both complexes, the linker sequences connecting the major and minor sites (residues 863–873 and 539–550 for RB and N1N2, respectively) have B-factors above the average number for the entire peptides (71.1 and 82.8 Å2 for RB and N1N2, respectively). By contrast, the residues bound to the major sites of both complexes have lower B-factors (40.5 and 47.6 Å2 for position P2–P5 of RB and N1N2, respectively), reflecting the strong interaction of these residues with the protein. There is 2573 and 2715 Å2 of surface area buried between m-Impα and the RB and N1N2 peptide, respectively. All residues of the RB peptide, except residues 865 and 869, and of the N1N2 peptide, except residues 542 and 546, make contacts with m-Impα at distances below 4 Å (Fig. 3). The minor and major site portions of the RB and N1N2 peptides have very similar structures (Fig. 4); after superposition of the equivalent Cα atoms, the r.m.s. deviation of the residues in positions P1–P6 is 0.19 Å, and the r.m.s. deviation of the residues in positions P1′–P3′ is 0.03 Å. By contrast, they adopt very different conformations in the linker regions between positions P3′ and P1; the path of the peptide chain is more linear in the case of RB than N1N2. The bipartite NLS linker sequence of both peptides makes favorable interactions with the H3 helices of armadillo repeats 4–7 of Impα. The most important contacts are limited to residues Asn868 and Lys871 of RB and Lys547 of N1N2. Among other interactions, the conserved residues Arg315 (Arm 6) and Tyr277 (Arm 5) of Impα that interrupt the regularity of the Trp-Asn array (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar) make extensive main chain and side chain contacts with the peptides. These residues also interact with nucleoplasmin NLS in mammalian and yeast Impα (10Conti E. Kuriyan J. Structure. 2000; 8: 329-338Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar). However, in that case, the interaction involves only the main chain of the peptide. Other important contacts are made with Arg238 (main chain of RB and N1N2), Ser276 (side chain of N1N2), and Ser234 (side chain of RB). Comparison with Other NLS Peptide-Importin-α Complex Structures—Significantly, the work presented here allows us for the first time to perform a detailed comparison of the structural determinants of binding of a number of NLSs to Impα, to align the NLSs with the binding pockets on Impα with some confidence, and to draw some general conclusions on the specificity of NLS binding (Table II). Despite diverse sequences, the binding of both basic clusters (positions P1–P5 and P1′–P2′) is similar in all the available structures, and the major differences occur in the linker regions connecting the basic clusters (Fig. 4). The exceptions are the N- and C-terminal portions of nucleoplasmin NLS bound to m-Impα, where the side chains of Lys155 (position P1′; N terminus of the peptide) and Lys170 (position P5; C terminus of the peptide) follow the direction of the main chain of the other peptides. Because all the other peptides contain at least one additional residue at the N and C termini, the conformation of the nucleoplasmin NLS peptide may be an artifact of the short length of the peptide. The case of nucleoplasmin highlights the importance of residues preceding position P1′ and following position P6.Table IIBinding of NLSs to specific binding pockets of importin-aNLS source proteinMinor NLS binding siteLinkerMajor NLS binding siteP1′P2′P3′P4′P1P2P3P4P5NucleoplasminKRPAATKKAGQAKKKKRBKRSAEGSNPPKPLKKLRN1N2RKKRKTEEESPLKDKAKKSKT-AgPKKKRKVPKKKRKc-MycKRVKLPAAKRVKConsensusKRX10-12KRXK Open table in a new tab The structures of nucleoplasmin NLS bound to y-Impα and m-Impα are significantly different (r.m.s. deviation of Cα atoms of residues 155–170 is 2.44 Å). In addition to the differences caused by the different lengths of the peptides discussed above, some differences may be explained by structural differences between the two Impα proteins; the yeast structure is slightly more “open” than the mouse structure (12Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (319) Google Scholar). Most of the differences are found in the region comprising residues 159–165 (there is high structural similarity when only the major (r.m.s. deviation of Cα atoms for positions P2–P5 is 0.28 Å) and minor (r.m.s. deviation of Cα atoms for positions P1′-P3′ is 0.24 Å) sites are superimposed). With the basic clusters binding most tightly, the different curvatures of the two Impα proteins appear to be compensated in the linker region. Although some linker region residues have different conformations, the main contacts of this region with Impα are comparable in both nucleoplasmin structures. The most important interactions occur for the main chain of conserved residues Arg315, Arg238, and Tyr277 of m-Impα (Arg321, Arg244, and Tyr283 of y-Impα). The portions of RB and N1N2 peptides bound in the major and minor sites superimpose closely with the nucleoplasmin NLS (the r.m.s. deviations of Cα atoms of positions P1–P5 and positions P1′–P3′ are 0.34 and 0.42 Å between RB and nucleoplasmin and 0.33 and 0.42 Å between N1N2 and nucleoplasmin, respectively). The structure of the nucleoplasmin linker region is more similar to that of RB than that of N1N2 (r.m.s. deviation of Cα atoms of linker region is 1.70 Å between RB (864–873) and nucleoplasmin (157–165) and 2.20 Å between N1N2 (541–550) and nucleoplasmin (157–165)), mainly in the region closest to the major site (residues 162–165 for nucleoplasmin). The region of the linker closest to the major site is structurally conserved best among the three bipartite NLS peptides. Binding of Bipartite NLS Linker Regions—The nucleoplasmin NLS-Impα complexes (10Conti E. Kuriyan J. Structure. 2000; 8: 329-338Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar) showed that the main chain of the linker region binds to the conserved Impα residues Arg315 (Arm 6) and Tyr277 (Arm 5) (Arg321 and Tyr283 for y-Impα; these residues interrupt the regularity of Trp-Asn array (11Fontes M.R.M. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar)). The conserved residue Arg238 (Arm 4) (Arg" @default.
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• W1995160911 title "Structural Basis for the Specificity of Bipartite Nuclear Localization Sequence Binding by Importin-α" @default.
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