Traffic 2013; 14: 1144–1154 © 2013 John Wiley & Sons A/S.Published by John Wiley & Sons Ltd

doi:10.1111/tra.12098

Distinctive Conformation of Minor Site-Specific NuclearLocalization Signals Bound to Importin-α

Chiung-Wen Chang1,2, Rafael Miguez

Counago1,2, Simon J. Williams1,2,

Mikael Boden1,3 and Bostjan Kobe1,2,∗

1School of Chemistry and Molecular Biosciences andInstitute for Molecular Bioscience, University ofQueensland, Brisbane, Qld 4072, Australia2Australian Infectious Diseases Research Centre,University of Queensland, Brisbane, Qld 4072, Australia3School of Information Technology and ElectricalEngineering, University of Queensland, Brisbane, Qld4072, Australia*Corresponding author: Bostjan Kobe,b.kobe@uq.edu.au.

Nuclear localization signals (NLSs) contain one or two

clusters of basic residues and are recognized by the

import receptor importin-α. There are two NLS-binding

sites (major and minor) on importin-α and the major NLS-

binding site is considered to be the primary binding site.

Here, we used crystallographic and biochemical methods

to investigate the binding between importin-α and

predicted ‘minor site-specific’ NLSs: four peptide library-

derived peptides, and the NLS from mouse RNA helicase

II/Guα. The crystal structures reveal that these atypical

NLSs indeed preferentially bind to the minor NLS-binding

site. Unlike previously characterized NLSs, the C-terminal

residues of these NLSs form an α-helical turn, stabilized

by internal H-bond and cation-π interactions between

the aromatic residues from the NLSs and the positively

charged residues from importin-α. This helical turn

sterically hinders binding at the major NLS-binding site,

explaining the minor-site preference. Our data suggest

the sequence RXXKR[K/X][F/Y/W]XXAF as the optimal

minor NLS-binding site-specific motif, which may help

identify novel proteins with atypical NLSs.

Key words: crystal structure, importin-alpha, nuclear

localization signal, nucleocytoplasmic transport, RNA

helicase II/Guα

Received 7 May 2013, revised and accepted for publi-

cation 31 July 2013, uncorrected manuscript published

online 2 August 2013, published online 26 August 2013

In eukaryotic cells, the transport of macromoleculesbetween the nucleus and cytoplasm occurs through thenuclear pore complexes (NPCs) in a highly regulated man-ner (1,2). The best understood pathway of nucleocytoplas-mic transport is the classical nuclear import pathway. Pro-teins destined for the nucleus contain targeting sequencestermed nuclear localization signals (NLSs). The classicalNLS (cNLS) is the best-characterized transport signal, andis comprised of either one (monopartite) or two (bipartite)

stretches of basic amino acids. The cNLSs are recognizedby the adaptor molecule importin-α (Impα), which linksthe cargo to the carrier molecule, importin-β (Impβ). Impβ

mediates the interaction of the trimeric complex with thenuclear pore as it translocates into the nucleus (3,4).

The classical monopartite and bipartite NLSs are exempli-fied by the NLSs from the simian virus 40 large T-antigen(SV40TAgNLS, P126KKKRRV132; the basic cluster is under-lined) and the Xenopus laevis protein nucleoplasmin(NplNLS, K155RPAATKKAGQAKKKK170), respectively (5,6).The crystal structures of cNLS-containing peptides andproteins bound to yeast, mouse and human Impα pro-teins show that cNLS-binding is highly conserved (7–10).Impα is composed of a flexible N-terminal Impβ-bindingdomain (IBB domain) and a cNLS-binding domain com-prised of ten armadillo (ARM) repeats. There are twodistinct NLS-binding sites on Impα, termed major (ARMrepeats 2–4) and minor (ARM repeats 6–8) NLS-bindingsites. Typical monopartite cNLSs are able to bind to bothsites, although the major site is considered to be thepreferential binding site (8,11,12). The bipartite cNLSsuse the larger basic cluster to bind to the major NLS-binding site, and the smaller N-terminal cluster to bindsimultaneously to the minor NLS-binding site. Structuraland interaction studies have dissected the binding ofcNLSs to Impα and established consensus sequencemotifs for both monopartite (K[K/R]X[K/R], X: any residue)and bipartite (KRX10-12K[K/R]X[K/R]) cNLSs (3,4,8,12–14).The core residues binding to the major site and theminor site are designated P1-P5 and P1’-P4’, respectively(4,8,11,12,15,16). A combination of mutagenesis and bio-physical studies dissected the differential contributionsmade to the interaction by residues at the different posi-tions within the cNLSs (4,15–19). A Lys at the P2 positionmakes a major contribution to the overall binding energy,followed by positions P3 and P5 occupied by Lys or Arg.Position P4 tolerates a greater variability of residues andmakes a smaller contribution to the binding energy. Theoptimal sequences for binding to the major NLS-bindingsite have been identified using oriented peptide libraryscreening (KKKRR, KKKRK and KKRKK for mouse Impα2,human Impα1 and human Impα5, respectively) (20). Thebinding cavities at the minor NLS-binding site are lesswell defined. A lysine-arginine (KR) motif is almost alwayspresent in the positions P1’ and P2’ of the minor sitein Impα:NLS structures characterized, and the preferencefor these residues is supported by systematic mutationalanalysis (19) (Table 1).

On the basis of the results of screening random peptidelibraries using mRNA display, Kosugi and colleaguessubdivided NLSs into six classes (24). Among the

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Table 1: NLS binding to the minor NLS-binding site in mImpα, based on structural information

NLS P-2’ P-1’ P0’ P1’ P2’ P3’ P4’ PDB ID (Reference)

SV40TAg K K R K V 1EJL (12)Guα S R G Q K R S F S K A F G Q 3ZIN (this study)A28 I G R K R G Y S V A F G G 3ZIO (this study)A58 W A G R K R T W R D A F 3ZIP (this study)B6 S S H R K R K F S D A F 3ZIQ (this study)B141 R V Q R K R K W S E A F 3ZIR (this study)Nup2p M A K R V A D A Q I . . . . . . . . . . . . 2C1T (21)hPLSCR4 G S I I R K W N 3Q5U (22)TPX2 G K R K H E . . . 3KND (23)Nucleoplasmin A V K R P A T K . . . 3UL1 (15)Bimax2 R R R K R K R E W . . . 3UKX (15)Class 3 K R X W/F/Y X X A F (24)Class 4 R/P X X K R K/R ˆDE (24)Consensus K/R X X K R X F/Y/W X X A F This study

The NLS sequences are aligned based on the interaction with mImpα minor NLS-binding site. The positions highlighted in bold (P1’ andP2’) correspond to the small basic cluster of a classical bipartite NLS. The positions P-2’ to P4’ are designated as in (15). The italicsindicate residues that have not been modeled in the crystal structures and ‘ . . . ’ indicates additional sequences not shown in the table.

six classes, two novel monopartite NLS consensussequences designated as class 3 (KRX[W/F/Y]XXAF) andclass 4 ([R/P]XXKR[K/R][ˆDE], where [ˆDE] represents anyresidues except for Asp or Glu) NLSs, were proposed to beminor site-specific (24). However, no structural data hasbeen available to support this binding mode. To date, twoNLSs that bind preferentially to the minor NLS-bindingsite have been characterized crystallographically: theNLS from human phospholipid scramblase 4 (hPLSCR4;G273SIIRKWN280) and the NLS from the mitotic regulatorprotein TPX2 (K284RKH287) (22,23). The hPLSCR4 andTPX2 NLSs contain only two and three basic residues,respectively, contributing limited interactions at the minorNLS-binding site.

A naturally occurring NLS from the C-terminus of mouseRNA helicase II (RH-II)/Guα (GuαNLS, K842RSFSKAF)resembles the class 3 minor site-specific NLSs. A C-terminal truncation of Guα resulted in a decrease of GFP-fused protein translocated into the nucleus in an in vivonuclear import assay (24). Guα is a multifunctional enzymethat possesses ATP-dependent double-stranded RNA-unwinding (5′ to 3′ direction) and RNA-folding activities.The unique NLS motif with only two basic residues (K842R),flanked C-terminally by two Phe residues, is distinct fromthe hPLSCR4 and TPX2 NLSs.

Here, using crystallographic and biochemical approaches,we investigated the binding of four peptide library-derivedclass 3 minor site-specific NLSs, and the naturally occur-ring GuαNLS, to mouse Impα (mImpα). The structuraldata demonstrate that these NLS peptides bind preferen-tially to the minor NLS-binding site, using a distinct bindingmode featuring an α-helical turn at the peptide C-terminus.This helical turn sterically hinders binding at the majorNLS-binding site, explaining the specificity for the minorNLS-binding site. The crystallographic results are sup-ported by comparing the binding affinities of the NLS

peptides to minor and major NLS-binding-site mutants ofmImpα. We also show that adding N-terminal residues tothe basic cluster in GuαNLS (S838RGQ) contributes signif-icantly to the binding affinity, and this is supported by theextensive interactions observed in the crystal structure.Together, our study dissects the binding cavities at theminor NLS-binding site of mImpα and elucidates the deter-minants of binding specificity for minor site-specific NLSs.

Results

N-terminal flanking residues contribute

to the binding affinity of GuαNLS for importin-α

We first determined, using microtiter-plate binding assays,the dissociation constants (Kd) for binding to mImpα

lacking the IBB domain (mImpα�IBB; the IBB domainwas removed to avoid competition for binding betweenthe autoinhibitory region and the NLS), of four class 3,minor site-specific NLS peptides, identified by Kosugiand colleagues based on peptide library experiments (24).The Kd (∼9 nM) measured for SV40TAgNLS, used as thepositive control, was comparable to the values reported inthe literature, using a variety of methods (17–19,25,26).There was no binding detected with GST alone (negativecontrol). The affinities measured for B6, B141, A28 andA58 NLS peptides ranged from 70 nM to 2.8 μM (Table 2,Figure 1). Unexpectedly, although the naturally occurringNLS from Guα (Guα*NLS, K842RSFSKAFGQ) was demon-strated to be functional using in vivo nuclear import assays(24), the corresponding peptide showed weak bindingaffinity (Kd = 5.2 μM), at the lower end of the affinityrange suggested for functional NLSs (∼10 nM to 1 μM)(28–32). Therefore, we included additional residues inthe N-terminal direction (S838RGQ); the extended peptide(GuαNLS, S838RGQKRSFSKAFGQ) showed a 100-foldincrease in binding affinity for mImpα�IBB. This observa-tion highlights that apart from the classical binding pockets

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Table 2: The dissociation constants (Kd) for mImpα�IBB:NLS interactions

Kd (μM) SV40TAg GuαNLS Guα*NLS A28 A58 B6 B141

mImpα�IBB 0.0090 ± 0.0006 0.082 ± 0.017 5.22 ± 0.76 0.200 ± 0.025 2.78 ± 0.70 0.060 ± 0.009 0.073 ± 0.012mImpα�IBBD192K 3.91 ± 0.27a 0.061 ± 0.02 ND 0.76 ± 0.16 3.8 ± 0.9 0.043 ± 0.006 0.050 ± 0.009mImpα�IBBE396R 0.034 ± 0.005a 7.13 ± 1.50 ND 4.70 ± 0.83 7.4 ± 2.0 2.40 ± 0.30 4.20 ± 0.59

The Kd values (in μM) were calculated using the program Graphpad (Prism). Each assay was performed in triplicate and the values withstandard error correspond to the best fit to the one-site-specific binding equation [Y = Bmax*X /(Kd + X ), Bmax is the maximum specificbinding with the same unit as Y , Kd is the equilibrium binding constant and X is ligand concentration]. GuαNLS: G838RGQKRSFSKAF849.Guα*NLS: K842RSFSKAF849. ND: not determined.aValues from (27).

Figure 1: Binding isotherms of minor site-specific NLS peptides binding to mImpαΔIBB. Impα concentration is depicted on thex-axis (shown as log values in the inset), and the absorbance at 450 nm is depicted on the y-axis. The plots were prepared with GraphPad(Prism) using nonlinear regression assuming one-site binding. Although there is more than one binding site, one site has a much higheraffinity than the other and one-site binding allows the comparison of overall binding affinities between different samples and with valuesreported in the literature (17,18,26). The mImpα�IBB_D192K and mImpα�IBB_E396R contain point mutations in the major and minorNLS-binding sites, respectively.

(binding P1’ and P2’ residues in the NLS), the C-terminalregion of mImpα provides additional binding cavities,consistent with what was observed for the high-affinitybipartite NLSs and plant-specific NLSs (15,19,27).

Structural basis of minor site-specific NLS binding

to importin-α

To define the structural basis of binding to Impα of minorsite-specific NLSs, we determined the crystal structuresof the A28, A58, B6 and B141 minor site-specific NLSpeptides in complex with mImpα�IBB, at resolutionsranging from 2.1 to 2.3 A (Table 3). A superposition ofthe four structures indicates that all the peptides use ananalogous binding mode (Figure 2A). All peptides bindwith the main-chain of the peptide running anti-parallel tothe direction of ARM repeats of mImpα, as observed forother Impα:NLS complexes (4). They all bind to both the

minor and major NLS-binding sites on mImpα, but morecontacts are observed and the crystallographic B-factors(indicating relative atomic displacements) are lower for thepeptides bound to the minor site (Table 4). These resultsare consistent with the minor NLS-binding site being thepredominant binding site for these peptides.

The electron density maps allow the modeling of the fullpeptide in the minor site for all four peptides (Figure 2B).Unexpectedly, all peptides exhibit an α-helical turn in theC-terminal region of the peptide (Figure 2A), which hasnot been observed in any other Impα:NLS structuresreported so far (4). In all the peptides, Lys and Arg occupy,respectively, the core P1’ and P2’ positions. The P1’ Lysforms H-bonds with Thr328, Asn361 and the main-chaincarbonyl of Val321. The P2’ Arg has its side-chain sand-wiched between the indole rings of Trp357 and Trp399,

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Table 3: Data collection and refinement statistics for mImpα:NLS crystals

Peptide complex A28 A58 B6 B141 GuαNLS

Space group P212121 P212121 P212121 P212121 P212121

a, b, c (A) 78.94 79.14 79.21 79.00 79.0990.18 90.18 90.21 90.34 90.26100.70 99.43 100.66 100.84 100.70

Data collectionResolution range (A)a 19.80–2.10 19.87–2.40 19.87–2.10 19.82–2.30 19.83–2.00

(2.21–2.10) (2.53–2.40) (2.21–2.10) (2.42–2.30) (2.11–2.00)Rmerge

b 0.152 (0.916) 0.101 (0.948) 0.135 (0.688) 0.126 (0.875) 0.055 (0.462)Total observations 307 751 (45 249) 205 437 (29 972) 308 237 (44 559) 229 053 (33 325) 721 866 (102 533)Unique observations 42 561 (6133) 28 437 (4066) 42 743 (6137) 32 737 (4711) 49 123 (7056)Completeness (%) 99.8 (100.0) 99.8 (100.0) 99.8 (99.8) 99.8 (100.0) 99.8 (99.3)Multiplicity 7.2 (7.4) 7.2 (7.4) 7.2 (7.3) 7.0 (7.1) 14.7 (14.5)Mean I/σ(I) 10.5 (2.2) 15.0 (2.1) 6.9 (2.9) 12.5 (2.3) 32.7 (6.5)RefinementRcrystal/Rfree (%)c 18.35/20.20 16.43/19.05 17.67/20.19 18.78/21.12 17.99/19.85Bond length RMSD (A) 0.010 0.010 0.010 0.010 0.013Bond angle RMSD (◦) 0.98 1.01 0.96 1.01 1.20Ramachandran plot (%)d

Favored 99.08 99.08 99.08 99.08 99.10Allowed 0.92 0.92 0.69 0.69 0.90Outliers 0 0 0.23 0.23 0

Number of residues modeledMajor NLS-binding site 6 6 6 6 6Minor NLS-binding site 9 9 10 10 13

aNumbers in parentheses refer to the statistics for the highest resolution shell.bRmerge =∑

hkl(∑

i(|Ihkl,i −<Ihkl>|))/∑hkl,i<Ihkl>, where Ihkl,i is the intensity of an individual measurement of the reflection with Millerindices h, k and l, and < Ihkl > is the mean intensity of that reflection. Calculated for I >−3σ(I).cRwork =�hkl(||Fobshkl|−|Fcalchkl||)/|Fobshkl|, where |Fobshkl| and |Fcalchkl| represent the observed and calculated structure factoramplitudes. Rfree is equivalent to Rwork but calculated using 5% of the reflections not used in refinement.dCalculated using Molprobity (33).

and forms a salt-bridge with Glu396. Gly, Thr or Lys can befound at the P3’ position (Table 1), although the long basicside-chain of Lys appears favored as it can form H-bondswith mImpα residues Thr322 and Asn283. The P4’ positionis occupied predominantly by an aromatic residue (Tyr,Trp or Phe), forming cation-π interactions (36) with mImpα

Arg315 (Figure 2C). The α-helical turn in the C-terminalregion of the peptides is formed using internal H-bondswithin the peptide and cation-π interactions between thetwo aromatic residues from the NLS peptides and Arg315

and Lys353 of mImpα�IBB. In the N-terminal direction,the Arg residue at the P0’ position interacts with theside-chain of Asn403 (Figure 2C).

In the major NLS-binding site, the peptides adopt aconventional binding mode with basic residues occupyingthe P2 and P3 positions; however, only six residues couldbe modeled for each peptide in this site (Table 3). The C-terminal helical turn is not observed and the correspondingresidues are disordered (Figure 2A).

Extensive interactions of GuαNLS in the minor

NLS-binding site

The naturally occurring GuαNLS resembles the peptidelibrary-derived class 3 minor site-specific NLSs, and hasbeen shown to be functional as it can facilitate GFP-Guα

fusion protein translocation into the nucleus (24), despitecontaining only a short basic cluster (K842R). The bindingaffinity (Kd = 82 nM, Table 2) is comparable to an opti-mal monopartite NLS peptide (KKKRR) binding to themajor NLS-binding site of mImp�IBB (20). To investigatethe structural basis of GuαNLS-binding to mImp�IBB,we determined its crystal structure at 2.0 A resolution(Table 3). The peptide binds to both the major and minorNLS-binding sites, although the electron density is clearerin the minor NLS-binding site, where the entire peptidecould be modeled (Figure 3A,B). Only six GuαNLS residues(Q841KRSFS) could be modeled at the major NLS-bindingsite. The observed contacts formed with mImp�IBB andthe crystallographic B-factors suggest that GuαNLS pre-dominantly binds to the minor NLS-binding site (Table 4).

Comparable to the peptide library-derived NLSs, theGuαNLS also displays an α-helical turn at its C-terminus(Figure 3B). The binding interactions mainly includeH-bonds and salt bridges. The short basic cluster (K842R)corresponds to positions P1’-P2’ (3,4,12) (Table 1). Ser844

at the P3’ position does not contact mImpα, while Phe845

at the P4’ position and Phe849 further C-terminally, interactwith mImpα Arg315 and Lys353, respectively, using cation-π interactions (Figure 3D) (36). The energetic contributionof the two cation-π interactions (predicted by CaPTURE)

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Figure 2: Structural basis of binding of minor site-specific NLS peptides, derived from peptide library experiments, to

mImpαΔIBB. A) Superposition of the mImpα�IBB:NLS complexes (mImpα in light blue; A28 in light green; A58 in dark green; B6 inred; B141 in orange; all in cartoon representation). The NLS peptides bind to both the minor and major NLS-binding sites, but the minorsite is the primary binding site. B) Simulated annealing omit electron density maps (contoured at 2σ, blue mesh) superimposed ontothe structure of A28 peptide in light green stick representation. The peptide positions P1’ to P4’ corresponding to residues K4RGYare labeled. The binding conformations of all the NLSs shown in stick representation in the bottom panel display an α-helical turn atthe C-terminus. C) Schematic illustration of the interactions between mImpα with the B6 peptide at the minor NLS-binding site. Thebasic side-chains (in blue) of Lys or Arg form salt bridges and electrostatic interactions with acidic residue Glu396 (in red) and smallhydrophobic residues (in green). The aliphatic portions of the basic chains interact with the Trp residues (green). Asn residues frommImpα (green) and main-chain amides from the peptide interact through hydrogen bonds (dotted lines). The aromatic side-chains of Pheresidues (orange) form cation-π interactions with positively charged (Lys and Arg) residues (blue) from mImpα. The residues shown inblack are from the B6 peptide.

Table 4: Properties of mImpα:NLS interactions based on the corresponding crystal structures

mImpα�IBB/NLS NHB NSB

Surface area buried inthe interface (A2)

Average B-factor(A2) of Impα�IBB

Average B-factor(A2) of NLS

mImpα�IBB/A28 9 (9) 2 (1) 1181.0 (1135.4) 34.73 36.07 (43.56)mImpα�IBB/A58 8 (8) 2 (1) 1255.8 (1221.6) 53.06 66.78 (69.40)mImpα�IBB/B6 8 (8) 2 (1) 1384.5 (1305.1) 36.75 42.14 (53.57)mImpα�IBB/B141 12 (11) 2 (1) 1578.1 (1354.9) 38.34 47.78 (68.14)mImpα�IBB/Guα 11 (9) 4 (1) 1378.1 (1109.0) 40.32 47.07 (41.31)

The numbers of hydrogen bonds (NHB), salt bridges (NSB) and the buried surface area (BSA) of the interface between Impα and NLSwere calculated using the program PISA (34). Values in parentheses correspond to the major NLS-binding site. Average B-factors formImpα and the NLS peptides, indicating relative atomic flexibilities, were calculated using program Baverage (35).

are −3.86 and −3.89 kcal/mol, and fall below the thresholdvalue of −2.00 kcal/mol value indicating a significantcation-π interaction (37). The main-chain atoms of Phe845,Ala848 and Phe849 residues facilitate the formation of theα-helical turn at the C-terminus of the peptide (Figure 3C).

The interactions by the N-terminal residues of GuαNLSaccount for the 100-fold increase in binding. Arg839,

Gly840 and Gln841 correspond to positions P-2’, P-1’ andP0’ (15). While the P-1’ Gly840 does not contact mImpα,the backbone carbonyl of the P0’ Gln841 interacts withthe polar amide side-chain of mImpα Asn403, while theP-2’ Arg839 forms a salt-bridge with the highly conservedmImpα Asp325. The side-chain hydroxyl oxygen of Ser838

at the P-3’ position forms H-bonds with the main-chaincarbonyl oxygen of the mImpα Ser406 (Figure 3C).

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Figure 3: GuαNLS shows extensive interactions at the minor NLS-binding site of mImpαΔIBB. A) Structure of GuαNLS in complexwith mImpα�IBB (in magenta and light blue cartoon representation, respectively) shows that GuαNLS forms an α-helical turn in theC-terminal region and preferentially binds to the minor NLS-binding site. B) Simulated annealing omit electron density maps (contouredat 2σ, blue mesh) superimposed onto the structure of GuαNLS in magenta stick representation. The peptide positions P-2’ to P4’corresponding to residues R839GQKRSF are labeled. A cartoon illustration of the GuαNLS conformation is shown in the bottom panel. C)Schematic illustration of the interactions between mImpα with the GuαNLS at the minor NLS-binding site. The basic side-chains (in blue)of Lys and Arg form salt bridges and electrostatic interactions with acidic residues (Asp and Glu in red). The aliphatic portions of thebasic chains interact with the Trp residues (green). Asn residues from mImpα (green) and main-chain amides from the peptide interactthrough hydrogen bonds (dotted lines). The aromatic side-chains of Phe residues (orange) form cation-π interactions with positivelycharged (Lys and Arg) residues (blue) from mImpα. The residues shown in black are from GuαNLS. D) Two aromatic residues (Phe845,Phe849) of the Guα/minor site-specific NLSs (in magenta stick/cartoon representations) are located in close proximity (3.6–4.4 A) to thebasic resides, Arg315 and Lys353, from mImpα�IBB. The Arg315 is also stabilized by the nearby residue, Glu354.

Mutational analysis confirms the minor site-specific

NLS-binding mode observed crystallographically

To verify the NLS-binding mode observed crystallographi-cally, the mImpα�IBB proteins containing point mutationsat either major or minor NLS-binding sites were used totest the effect on the binding of minor site-specific NLSs.The residues Asp192 and Glu396 (Asp203 and Glu402 in yeastImpα) are essential for NLS-binding to the major and minorNLS-binding sites, respectively (17,38,39). In our assays,the prototypical SV40TagNLS bound to the mImpα�IBB

minor-site mutant (E396R) with higher affinity than themajor-site mutant (D192K) (Table 2). By contrast, the minorsite-specific NLS peptides, including GuαNLS, A28, A58,B6 and B141, bound to the mImpα�IBB major-site mutant(D192K) with higher affinity than the minor-site mutant(E396R). The mutation in the minor site of mImpα�IBB(E396R) caused a significant reduction in binding comparedto the wild-type mImpα�IBB, in particular for the GuαNLS,B6 and B141 peptides. The results are consistent with theNLS-binding mode observed in the crystal structures.

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Figure 4: Binding of minor site-specific NLSs to Impα. For clarity, mImpα�IBB has been omitted in all the panels and only thepeptides are shown (A) Left: Superposition of GuαNLS (magenta) and four minor site-specific NLS peptides (A28: light green; A58:dark green; B6: red; B141: orange) at the minor NLS-binding site shows the peptides use comparable binding modes. The minor-sitepositions P-2’ to P4’ are indicated. Right: as in the left panel, but for the major NLS-binding site. Only a part of each peptide is orderedin this case (positions P1-P6). (B) Superposition of mImpα:hPLSCR4 NLS (PDB ID: 3Q5U (22)), mImpα:TPX2 NLS (PDB ID:3KND (23)),and mImpα:GuαNLS (this study) complex structures [root mean square distances (RMSD): 0.253 and 0.245 A for 380 and 406 Cα atomsof mImpα, and 1.46 and 0.43 A for 4 Cα atoms of NLSs, for the hPLSCR4 NLS and TPX2 NLS complexes compared to the GuαNLScomplex, respectively]. GuαNLS has the most extensive interactions with mImpα.

Bioinformatic analysis indicates that minor-site

specific NLSs are much less prevalent than the

classical NLSs

To evaluate the occurrence of minor-site-specific NLSs innuclear proteins, we computationally surveyed the mouseproteome. Using sequence alignments of class 3 and class4 minor-site-specific NLSs provided in the literature (24),we first constructed position-weight matrices (PWMs) ofeach (40). We collated all mouse proteins that can beconfidently assigned to belong to the nuclear proteome(positives), and a similar-sized set of proteins that wereconfidently non-nuclear (negatives). We then scored allproteins at each sequence position using these PWMsto identify candidate binding sites. Both classes showno statistically significant enrichment in the positive set(class 3: p = 0.42; class 4: p = 0.30). When combining thesequences from both classes into the same alignment,a slight but not statistically significant enrichment in thepositive set was observed (p = 5.3e−02). As a control,by using the alignment of class 1 classical monopartiteNLS sequences (again from (24)), we observed a clearlysignificant enrichment among nuclear as opposed to non-nuclear proteins (p = 1.4e−04, 51 matches in the positiveset). The result indicates that minor-site-specific NLSs aremuch less prevalent than the classical NLSs.

Discussion

A novel binding conformation for minor site-specific

NLSs

The identification of atypical NLSs binding specifically tothe minor NLS-binding site of Impα (22–24,27) highlightsthat the minor NLS-binding site functions not only to allowthe binding of the N-terminal basic cluster of bipartiteNLSs, but can also serve as the primary binding sitefor certain cargo proteins. To understand the structuralbasis of the binding of atypical NLSs, we determined thecrystal structures of mImpα�IBB in complex with fourpeptide library-derived minor site-specific NLS peptides,as well as the naturally occurring NLS from the mouseRNA helicase II protein, Guα. In accord with suggestionsby Kosugi et al. (24), our structural and mutationalanalyses show that these NLSs bind preferentially to theminor NLS-binding site in Impα. Unexpectedly, these NLSpeptides present a novel binding conformation with anα-helical turn formed in the C-terminal region (Figure 4A),which is unique among the Impα:NLS complex structuresreported so far (4,7,8,12,15,20,22,23,27,41,42), and alsothe primary reason for the preference for the minorNLS-binding site. To date, the majority of monopartiteNLSs have been found to bind in an extended

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conformation and preferentially to the major NLS-bindingsite. Two NLSs, from hPLSCR4 and TPX2 proteins, havebeen previously shown by crystallographic methods tobind to the minor NLS-binding site, but the interactionsthey form with Impα are limited (including only ARMrepeats 7–8). In particular, the affinity for hPLSCR4 NLS(Kd = 48.7 μM)) is weaker than any of the minor site-specific NLS peptides studied here (Table 2), althoughit is active in nuclear import in the context of thePLSCR4 protein, or fused to a reporter protein, both inpermeabilized and live cells (22). Both hPLSCR4 and TPX2NLSs possess a lower content of basic residues and havea ring in the P4’ residue (His287 of TPX2 NLS, Trp279 ofhPLSCR4 NLS); these observations were suggested asreasons for the reduced affinity for the major NLS-bindingsite (23). Superposition of both NLSs onto GuαNLS showsa comparable binding conformation for the backbone ofresidues in positions P1’-P4’ (Figure 4B), whereas GuαNLShas more extensive contacts than the other two NLSs atposition P-2’. While an aromatic residue (F/Y/W) is presentin the P4’ position in all of the peptides studied here,the side-chain is oriented differently and forms cation-πinteractions with mImpα Arg315. This interaction mimicsthe binding of a basic residue in the P5 position to Trp142

and Trp184 of the major NLS- binding site (18,23), andthe specific recognition of IBB domain by Imp-β (43).The α-helical turn forms beyond the P4’ pocket; if wesuperimpose the peptide in this conformation into themajor NLS-binding site, the α-helical turn shows stericclashes with the N-terminal region of Impα (ARM repeats1–2) (Figure 5). The binding conformation may thereforebe in large part responsible for the specificity of theseNLSs for the minor NLS-binding site.

Optimal minor NLS-binding site-specific sequence

Table 1 compares the binding of all NLSs studied herewith other selected NLSs, relative to the binding pocketsin the minor NLS-binding groove of mImpα. The best-defined pockets (P1’and P2’ residue-binding pockets)are exclusively occupied by Lys and Arg, respectively,except for an Ile residue from the hPLSCR4 NLS atthe P1’ position. An Arg at the P2’ position appears tobe the residue with the most important contribution tothe binding energy (15,22). Comparison of the bindingaffinities of the peptide library-derived peptides (Table 2)shows that a Lys residue at the P3’ position correlates withstronger binding, consistent with previous observations(15,24). The minor site-specific NLS peptides further utilizea large aromatic side-chain at the P4’ position to formcation-π interactions with mImpα Arg315. All the peptidescontain a Phe residue three residues C-terminal to P4’,which forms similar cation-π interactions with mImpα

Lys353, in addition to the internal H-bond interactions withthe P4’ position of the peptide. The two aromatic residuestherefore mediate the formation of the specific bindingconformation at the minor NLS-binding site. Interestingly,the residues at positions P-2’ to P0’ make a significantcontribution to binding of GuαNLS, but do not make anysignificant contacts in the peptide library-derived peptides.

Figure 5: The α-helical turn prevents the minor site-specific

NLSs binding to the major NLS-binding site. Top panel: ThemImpα�IBB:GuαNLS structure is shown with the molecularsurface from mImp�IBB in light blue and the GuαNLS in magentacartoon representation. Bottom panel: superposition of GuαNLSfrom the minor NLS-binding site onto the partial GuαNLS segmentat the major NLS-binding site shows a steric clash (magnified atthe right-hand corner) between the GuαNLS and N-terminal regionof mImpα�IBB. The top and bottom images are related by a 90◦

rotation around the x-axis.

The P-2’ Arg in GuαNLS forms similar interactions tothose seen for the analogous position in the high-affinitybipartite NLS peptide Bimax2 (15). This position containseither Arg or Pro in the consensus sequence of theclass 4 minor-site-specific NLSs ([R/P]XXKR[K/R][ˆDE])(24). Although this group of minor site-specific NLSs hasnot been characterized structurally, some of the bindingdeterminants can be explained by the available structuraldata. The binding of the class 4 minor NLSs appears to beweaker than the class 3 minor NLS to yeast Impα by thein vitro binding assay (24).

Combining all the available results, our studies allowus to propose the sequence [K/R]XXKRX[F/Y/W]XXAFas the optimal minor NLS-binding site-specific motif(Table 1). However, this sequence is not enriched inthe mouse nuclear proteome (p = 0.46), similar to whatwe observed above for the class 3 and class 4 minorsite-specific NLSs described in the literature. This implies

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that the minor site-specific NLS motif is not a strongindicative feature for a nuclear protein, unlike the classicalNLS. Unlike the optimal major NLS-binding site-specificsequences that contain a cluster of five positively chargedresidues and bind in an extended conformation (20), theminor site-specific NLSs contain only a two-residue basiccluster (KR) and use a more complex conformation forbinding.

Conclusions

In summary, our study illustrates the molecular andstructural basis of binding determinants of minor site-specific NLSs, identified originally by Kosugi et al.(24). The crystallographic data reveal the novel bindingconformation of the minor site-specific NLSs and explainthe reasons for preferential binding to the minor NLS-binding site. We show that the minor NLS-binding sitecan substitute the major NLS-binding site as a primarybinding site for certain cargo proteins, and allow nM-rangeaffinity binding. Our work highlights that the extendedbinding surface of Impα, based on ARM-repeat scaffold,can accommodate diverse NLS sequences. Our workallows us to define the optimal determinants of thebinding sequence of the minor site-specific NLSs andthe extended binding cavities at the minor NLS-bindingsite, which may help identify novel proteins destined tothe nucleus that contain atypical NLSs (40).

Materials and Methods

Generation of recombinant DNA constructsThe cDNAs corresponding to the NLSs, containing BamHI and EcoRIrestriction sites at the 5′ end and 3′end, respectively, were clonedinto the pGEX2T vector (GE Healthcare) pre-treated with restrictionenzymes (BamHI-HF and EcoRI-HF, 20 U/μL, NEB). The complementaryoligonucleotides were ligated into the pGEX2T vector using Quick T4DNA Ligase (New England Biolabs, NEB) according to the manufacturer’sinstructions.

Recombinant protein expression and purificationMouse Impα lacking the IBB domain (mImpα�IBB, residues 70–529;α2 isoform, NP_034785) and its mutants (mImpα�IBBD192K andmImpα�IBBE396R) were expressed as previously described (12), andpurified using a HisTrap column (5 mL; GE Healthcare), followed bysize exclusion chromatography (S-200, GE Healthcare) in 20 mM TrispH 7.8, 125 mM NaCl. The purification of mImpα�IBB:NLS complexeswas performed as previously described (15). The NLS peptides wereoverexpressed as GST (glutathione-S-transferase)-fusion proteins (GST-NLSs) using the autoinduction method (44) and immobilized on a GSTrapcolumn (5 mL; GE Healthcare). The column was further washed with10 column volumes of the binding buffer (50 mM Tris pH 7.8, 125 mM

NaCl) and injected with purified mImpα�IBB. The mImpα�IBB:GST-NLScomplex was eluted in the elution buffer (binding buffer containing10 mM glutathione) and digested with thrombin at 4◦C overnight.The proteins were further purified using S-200 and GSTrap columns.The pure mImpα�IBB:NLS complexes were concentrated to between11 and 18 mg/mL (Amicon filter, MWCO 10 kDa, Millipore). Theconcentrated proteins were snap-frozen in liquid nitrogen in smallaliquots.

Microtiter-plate binding assayThe solid-phase binding assay was performed essentially as previouslydescribed (15,41,45,46). The assay was carried out on Immuno MaxiSorp96-well plate (Thermo Fisher Scientific). The plates were coated with50 nM GST-NLS or GST for 16 h at 4◦C in the coating buffer [PBSsupplemented with 2 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride(PMSF)]. The plates were then washed three times with PBS and incubatedovernight at 4◦C in the binding buffer (coating buffer supplemented with3% BSA and 0.1% Tween). Binding reactions were carried out for 2 hat 4◦C with 100 μL/well of S-tagged mImpα�IBB in binding buffer. Afterbinding, plates were washed three times with binding buffer withoutBSA and proteins were incubated in the cross-linking buffer [1 mg/mL1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in the same buffer]for 15 min. The plates were then washed for 20 min in PBS-T (PBS and0.2% Tween 20), 10 min with PBS-T containing 100 mM ethanolamine,and finally incubated for 10 min in PBS-T containing 3% BSA. Afterwashing, the plates were incubated in S-protein-horseradish peroxidaseconjugate (Novagen) in the coating buffer containing 1% BSA and 0.1%Tween 20 for 1 h at 4◦C. After incubation the plates were washed threetimes by immersion in PBS. Horseradish peroxidase substrate (100 μg/mL3,3′,5,5′-tetramethylbenzidine, Sigma) was then added for 10 min at roomtemperature and the reaction was stopped by adding an equal volumeof 0.5 M H2SO4. The signal was determined at 450 nm with a MolecularDevices plate reader (Spectra Max 250). GraphPad (Prism) was used toanalyze the binding data using nonlinear regression.

Protein crystallization and structure determinationThe crystallization conditions for the mImpα�IBB:NLS complexes werescreened starting from the conditions reported in the literature (7):0.4–0.9 M sodium citrate, 0.1 M HEPES buffer (pH 6.5–8.0) and 10 mM

DTT, using the vapor diffusion (hanging drop) method in 24-well Linbroplates at 18◦C. Single crystals were cryo-protected in the reservoir solutionsupplemented with 24% glycerol, and flash-cooled in liquid nitrogen. X-ray diffraction data were collected using beamline MX2 at AustralianSynchrotron with a CCD detector (ADSC Quantum 210r). The data wereprocessed using XDS (47) and further analyzed with programs from theCCP4 suite (35). The structures were determined by molecular replacementusing Morlep (35) and mImpα (PDB ID: 1IAL; (7)) with the autoinhibitorysegment omitted as the search model. Initial solutions were subjectedto rigid-body refinement using Refmac (35) and further refined usingPHENIX and BUSTER (48,49). The NLSs were built manually using theprogram Coot (50). The final structures were validated with MolProbity(33). Crystallographic data are summarized in Table 3.

Position weight matrix method-based proteome

screen and Fisher’s exact testThree alignments, containing 21, 15 and 6 peptides, were adopted from theliterature (24) to represent class 1 classical NLSs, and class 3 and class 4minor-site-specific NLSs, respectively. The PWMs were constructed fromthese alignments as described previously (27). The protein sequence datacorrespond to nuclear/non-nuclear protein sets from mouse, verified byobserving cellular localization of green fluorescence protein (GFP)-taggedproteins (3,4). There are 3336 sequences in the nuclear protein set and3933 sequences in non-nuclear protein set. Under the same threshold,proteins that have at least one predicted sequence motif were consideredpositives. All remaining proteins were considered negatives. Using thePWM method, the number of predicted positives is obtained for both thenuclear and non-nuclear protein sets. The one-tailed Fisher’s exact test(51) was used to assign a p-value to indicate the statistical significanceof the absolute prevalence of the motif in the mouse nuclear protein set,relative to the number of non-nuclear proteins containing the same motif.

Accession numbersThe atomic coordinates and structure factors have been deposited in theProtein Data Bank (mImpα:A28, 3ZIO; mImpα:A58, 3ZIP; mImpα:B6, 3ZIQ;mImpα:B141,3ZIR; mImpα:GuαNLS, 3ZIN).

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Acknowledgments

We thank the Australian Synchrotron beamline scientists for help with X-ray data collection; and Daniel J. Ericsson, Mary Marfori and the membersof Kobe lab for valuable suggestions. We acknowledge the use of theUniversity of Queensland Remote Operation Crystallization and X-ray (UQROCX) Diffraction Facility and the assistance of Karl Byriel and GordonKing. B. K. is a National Health and Medical Research Council ResearchFellow.

References

1. Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, DevosD, Suprapto A, Karni-Schmidt O, Williams R, Chait BT, Sali A, RoutMP. The molecular architecture of the nuclear pore complex. Nature2007;450:695–701.

2. Tran EJ, Bolger TA, Wente SR. SnapShot: nuclear transport. Cell2007;131:420.

3. Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, CorbettAH. Classical nuclear localization signals: definition, function, andinteraction with importin alpha. J Biol Chem 2007;282:5101–5105.

4. Marfori M, Mynott A, Ellis JJ, Mehdi AM, Saunders NF, Curmi PM,Forwood JK, Boden M, Kobe B. Molecular basis for specificity ofnuclear import and prediction of nuclear localization. Biochim BiophysActa 2011;1813:1562–1577.

5. Kalderon D, Richardson WD, Markham AF, Smith AE. Sequencerequirements for nuclear location of simian virus 40 large-T antigen.Nature 1984;311:33–38.

6. Dingwall C, Robbins J, Dilworth SM, Roberts B, Richardson WD. Thenucleoplasmin nuclear location sequence is larger and more complexthan that of SV-40 large T antigen. J Cell Biol 1988;107:841–849.

7. Teh T, Tiganis T, Kobe B. Crystallization of importin alpha, thenuclear-import receptor. Acta Crystallogr D Biol Crystallogr 1999;55(Pt2):561–563.

8. Conti E, Uy M, Leighton L, Blobel G, Kuriyan J. Crystallographicanalysis of the recognition of a nuclear localization signal by thenuclear import factor karyopherin alpha. Cell 1998;94:193–204.

9. Tarendeau F, Boudet J, Guilligay D, Mas PJ, Bougault CM, BouloS, Baudin F, Ruigrok RW, Daigle N, Ellenberg J, Cusack S, SimorreJP, Hart DJ. Structure and nuclear import function of the C-terminaldomain of influenza virus polymerase PB2 subunit. Nat Struct MolBiol 2007;14:229–233.

10. Dias SM, Wilson KF, Rojas KS, Ambrosio AL, Cerione RA. Themolecular basis for the regulation of the cap-binding complex bythe importins. Nat Struct Mol Biol 2009;16:930–937.

11. Conti E, Kuriyan J. Crystallographic analysis of the specific yet versatilerecognition of distinct nuclear localization signals by karyopherin alpha.Structure 2000;8:329–338.

12. Fontes MR, Teh T, Kobe B. Structural basis of recognitionof monopartite and bipartite nuclear localization sequences bymammalian importin-alpha. J Mol Biol 2000;297:1183–1194.

13. Chelsky D, Ralph R, Jonak G. Sequence requirements for syntheticpeptide-mediated translocation to the nucleus. Mol Cell Biol1989;9:2487–2492.

14. Dingwall C, Laskey RA. Nuclear targeting sequences – a consensus?Trends Biochem Sci 1991;16:478–481.

15. Marfori M, Lonhienne TG, Forwood JK, Kobe B. Structural basis ofhigh-affinity nuclear localization signal interactions with importin-alpha.Traffic 2012;13:532–548.

16. Fontes MR, Teh T, Jans D, Brinkworth RI, Kobe B. Structural basisfor the specificity of bipartite nuclear localization sequence binding byimportin-alpha. J Biol Chem 2003;278:27981–27987.

17. Leung SW, Harreman MT, Hodel MR, Hodel AE, Corbett AH.Dissection of the karyopherin alpha nuclear localization signal(NLS)-binding groove: functional requirements for NLS binding. JBiol Chem 2003;278:41947–41953.

18. Hodel MR, Corbett AH, Hodel AE. Dissection of a nuclear localizationsignal. J Biol Chem 2001;276:1317–1325.

19. Kosugi S, Hasebe M, Entani T, Takayama S, Tomita M, Yanagawa H.Design of peptide inhibitors for the importin alpha/beta nuclear importpathway by activity-based profiling. Chem Biol 2008;15:940–949.

20. Yang SN, Takeda AA, Fontes MR, Harris JM, Jans DA, Kobe B. Probingthe specificity of binding to the major nuclear localization sequence-binding site of importin-alpha using oriented peptide library screening.J Biol Chem 2010;285:19935–19946.

21. Matsuura Y, Stewart M. Nup50/Npap60 function in nuclear proteinimport complex disassembly and importin recycling. EMBO J2005;24:3681–3689.

22. Lott K, Bhardwaj A, Sims PJ, Cingolani G. A minimal nuclearlocalization signal (NLS) in human phospholipid scramblase 4 thatbinds only the minor NLS-binding site of importin alpha1. J Biol Chem2011;286:28160–28169.

23. Giesecke A, Stewart M. Novel binding of the mitotic regulator TPX2(target protein for Xenopus kinesin-like protein 2) to importin-alpha. JBiol Chem 2010;285:17628–17635.

24. Kosugi S, Hasebe M, Matsumura N, Takashima H, Miyamoto-SatoE, Tomita M, Yanagawa H. Six classes of nuclear localization signalsspecific to different binding grooves of importin alpha. J Biol Chem2009;284:478–485.

25. Catimel B, Teh T, Fontes MR, Jennings IG, Jans DA, HowlettGJ, Nice EC, Kobe B. Biophysical characterization of interactionsinvolving importin-alpha during nuclear import. J Biol Chem2001;276:34189–34198.

26. Chen MH, Ben-Efraim I, Mitrousis G, Walker-Kopp N, Sims PJ,Cingolani G. Phospholipid scramblase 1 contains a nonclassical nuclearlocalization signal with unique binding site in importin alpha. J BiolChem 2005;280:10599–10606.

27. Chang CW, Counago RL, Williams SJ, Boden M, Kobe B.Crystal structure of rice importin-alpha and structural basis of Itsinteraction with plant-specific nuclear localization signals. Plant Cell2012;24:5074–5088.

28. Rihs HP, Jans DA, Fan H, Peters R. The rate of nuclear cytoplasmicprotein transport is determined by the casein kinase II site flankingthe nuclear localization sequence of the SV40 T-antigen. EMBO J1991;10:633–639.

29. Hubner S, Xiao CY, Jans DA. The protein kinase CK2 site(Ser111/112) enhances recognition of the simian virus 40 largeT-antigen nuclear localization sequence by importin. J Biol Chem1997;272:17191–17195.

30. Hu W, Jans DA. Efficiency of importin alpha/beta-mediated nuclearlocalization sequence recognition and nuclear import. Differential roleof NTF2. J Biol Chem 1999;274:15820–15827.

31. Hu W, Kemp BE, Jans DA. Kinetic properties of nuclear transportconferred by the retinoblastoma (Rb) NLS. J Cell Biochem2005;95:782–793.

32. Hodel AE, Harreman MT, Pulliam KF, Harben ME, Holmes JS,Hodel MR, Berland KM, Corbett AH. Nuclear localization signalreceptor affinity correlates with in vivo localization in Saccharomycescerevisiae. J Biol Chem 2006;281:23545–23556.

33. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, WangX, Murray LW, Arendall WB 3rd, Snoeyink J, Richardson JS,Richardson DC. MolProbity: all-atom contacts and structure validationfor proteins and nucleic acids. Nucleic Acids Res 2007;35(Web Serverissue):W375–W383.

34. Krissinel E, Henrick K. Inference of macromolecular assemblies fromcrystalline state. J Mol Biol 2007;372:774–797.

35. CCP4. The CCP4 suite: programs for protein crystallography. ActaCrystallogr D Biol Crystallogr 1994;50:760–763.

36. Dougherty DA. Cation-pi interactions in chemistry and biology: a newview of benzene, Phe, Tyr, and Trp. Science 1996;271:163–168.

37. Gallivan JP, Dougherty DA. Cation-pi interactions in structural biology.Proc Natl Acad Sci U S A 1999;96:9459–9464.

38. Robbins J, Dilworth SM, Laskey RA, Dingwall C. Two interdepen-dent basic domains in nucleoplasmin nuclear targeting sequence:identification of a class of bipartite nuclear targeting sequence. Cell1991;64:615–623.

39. Efthymiadis A, Shao H, Hubner S, Jans DA. Kinetic characterizationof the human retinoblastoma protein bipartite nuclear localizationsequence (NLS) in vivo and in vitro. A comparison with the SV40 largeT-antigen NLS. J Biol Chem 1997;272:22134–22139.

40. Kobe B, Boden M. Computational modelling of linear motif-mediatedprotein interactions. Curr Top Med Chem 2012;12:1553–1561.

Traffic 2013; 14: 1144–1154 1153

Chang et al.

41. Takeda AA, de Barros AC, Chang CW, Kobe B, Fontes MR. Structuralbasis of importin-alpha-mediated nuclear transport for Ku70 and Ku80.J Mol Biol 2011;412:226–234.

42. de Barros AC, Takeda AA, Chang CW, Kobe B, Fontes MR. Structuralbasis of nuclear import of flap endonuclease 1 (FEN1). Acta CrystallogrD Biol Crystallogr 2012;68(Pt 7):743–750.

43. Koerner C, Guan T, Gerace L, Cingolani G. Synergy of silent andhot spot mutations in importin beta reveals a dynamic mechanismfor recognition of a nuclear localization signal. J Biol Chem2003;278:16216–16221.

44. Studier FW. Protein production by auto-induction in high densityshaking cultures. Protein Expr Purif 2005;41:207–234.

45. Lange A, McLane LM, Mills RE, Devine SE, Corbett AH. Expanding thedefinition of the classical bipartite nuclear localization signal. Traffic2010;11:311–323.

46. Matsuura Y, Lange A, Harreman MT, Corbett AH, Stewart M.Structural basis for Nup2p function in cargo release and karyopherinrecycling in nuclear import. EMBO J 2003;22:5358–5369.

47. Kabsch W. XDS. Acta Crystallogr D Biol Crystallogr 2010;66(Pt2):125–132.

48. Smart OS, Womack TO, Flensburg C, Keller P, Paciorek W, Sharff A,Vonrhein C, Bricogne G. Exploiting structure similarity in refinement:automated NCS and target-structure restraints in BUSTER. ActaCrystallogr D Biol Crystallogr 2012;68(Pt 4):368–380.

49. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, EcholsN, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoyAJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, et al.PHENIX: a comprehensive Python-based system for macromolecularstructure solution. Acta Crystallogr D Biol Crystallogr 2010;66(Pt 2):213–221.

50. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features anddevelopment of Coot. Acta Crystallogr D Biol Crystallogr 2010;66(Pt4):486–501.

51. Fisher RA. On the interpretation of x(2) from contingency tables, andthe calculation of P. J R Stat Soc 1922;85:87–94.

1154 Traffic 2013; 14: 1144–1154