Crystal structure of importin-a bound to a peptide bearing the nuclear localisation signal from chloride intracellular channel protein Andrew V Mynott1, Stephen J Harrop1, Louise J Brown2, Samuel N Breit3, Bostjan Kobe4,5 and Paul M G Curmi1,3 School of Physics, University of New South Wales, Sydney, NSW, Australia Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia St Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital and University of New South Wales, Sydney, NSW, Australia School of Chemistry and Molecular Biosciences and Centre for Infectious Disease Research, University of Queensland, Brisbane, Qld, Australia Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld, Australia Keywords chloride intracellular channel protein; CLIC4; importin-a; nuclear localization signal (NLS); nucleocytoplasmic transport Correspondence P Curmi, School of Physics, University of New South Wales, Sydney, NSW 2052, Australia Fax: +61 9385 6060 Tel: +61 9385 4552 E-mail: P.Curmi@unsw.edu.au (Received 17 November 2010, revised 31 January 2011, accepted 23 February 2011) doi:10.1111/j.1742-4658.2011.08086.x It has been reported that a human chloride intracellular channel (CLIC) protein, CLIC4, translocates to the nucleus in response to cellular stress, facilitated by a putative CLIC4 nuclear localization signal (NLS) The CLIC4 NLS adopts an a-helical structure in the native CLIC4 fold It is proposed that CLIC4 is transported to the nucleus via the classical nuclear import pathway after binding the import receptor, importin-a In this study, we have determined the X-ray crystal structure of a truncated form of importin-a lacking the importin-b binding domain, bound to a CLIC4 NLS peptide The NLS peptide binds to the major binding site in an extended conformation similar to that observed for the classical simian virus 40 large T-antigen NLS A Tyr residue within the CLIC4 NLS makes surprisingly favourable interactions by forming side-chain hydrogen bonds to the importin-a backbone This structural evidence supports the hypothesis that CLIC4 translocation to the nucleus is governed by the importin-a nuclear import pathway, provided that CLIC4 can undergo a conformational rearrangement that exposes the NLS in an extended conformation Database Structural data are available in the protein Data Bank under the accession number 3OQS Structured digital abstract CLIC4 and importin alpha bind by x-ray crystallography (View interaction) l Introduction The importin-a:b nuclear import pathway is one of the best understood nuclear trafficking systems in the cell [1] The pathway operates via the importin-a receptor, an armadillo (ARM) repeat protein, that recognizes and binds directly to cargo protein in the cytoplasm The importin-a:importin-b:cargo complex travels through the nuclear pore, with importin-b primarily responsible for negotiating passage through the nuclear pore complex This transport process is dependent on the ability of importin-a to recognize specific nuclear localization signals (NLSs) presented by the cargo protein The acidic environment of the importin-a binding sites confers a high Abbreviations ARM, armadillo; CLIC, chloride intracellular channel; NLS, nuclear localization signal; RSCC, real space correlation coefficient; TAg, simian virus 40 (SV40) large T-antigen 1662 FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS A V Mynott et al affinity to clusters of basic residues in the NLS Monopartite NLSs consist of a single cluster of basic amino acids, approximately six residues long, which generally interact with the major binding site in importin-a Structural studies have shown that an NLS binds importin-a in an extended conformation, suggesting that functional NLSs need to be unfolded and flexible within the cargo protein Recent studies have demonstrated an interaction between importin-a and the chloride intracellular channel (CLIC) protein, CLIC4 [2,3] The structure of a soluble form of CLIC4 shows that it adopts the canonical glutathione S-transferase fold with an N-terminal thioredoxin-like domain and an a-helical C-terminal domain [4] CLIC4 can form poorly selective anion channels that are sensitive to redox conditions [5] and, like other CLIC family members, it is hypothesized that CLIC4 undergoes a structural transition from the soluble form to an integral membrane form CLIC4 is functionally important in the cell and has recently been implicated in angiogenesis, with the observation that suppressed CLIC4 expression leads to the disruption of tubular morphogenesis [6] CLIC4 is upregulated in human and mouse differentiating cells [7] and has also been implicated in the regulation of tumour growth [8,9] In the event of cellular stress, CLIC4 translocates to the nucleus in human osteosarcoma cells as well as mouse S1 keratinocytes, where it is involved in an apoptosis pathway independent of the apoptotic protease activating factor [2] After translocation, CLIC4 localizes near the nuclear envelope and in the nucleoplasm Immunoprecipitation experiments have shown that tumour necrosis factor-a or etoposide treatment of keratinocytes increases the constitutive interaction between CLIC4 and various members of the nuclear import machinery, including Ran, nuclear transport factor-2 and importin-a [2] Mutagenesis of a cluster of basic residues in the putative CLIC4 NLS site (199KVVAKKYR206 to 199TVVAITYG206) is sufficient to prevent nuclear translocation, suggesting that this monopartite NLS-like sequence has an active role in the nuclear import process [2] This indicates that the binding of CLIC4 to importin-a via this putative NLS is responsible for nuclear translocation; however, in the crystal structure of soluble CLIC4, the putative NLS adopts a helical conformation that would preclude binding to importin-a (refer to Fig 2D) More recently, it has been shown that CLIC4 nuclear translocation is induced in mouse S1 keratinocytes by treatment with nitric oxide [3] The nuclear translocation is accompanied by S-nitrosylation of a Cys residue in CLIC4, Cys234 The S-nitrosylation of CLIC4 has been found to induce a conformational Crystal structure of importin-a:CLIC4 NLS peptide change which destabilizes the native conformation Such a destabilization may facilitate the interaction between the otherwise helical CLIC4 NLS and importin-a It has been shown that S-nitrosylation of CLIC4 enhances the interaction with importin-a, as determined by immunoprecipitation [3] In this article, we present the X-ray crystal structure of mouse importin-a (70–529) bound to a peptide corresponding to the CLIC4 NLS The importin-a (70– 529) construct used to obtain the importin-a:CLIC4 NLS complex lacks the first 69 residues that correspond to the flexible importin-b binding domain The importin-b binding domain is known to have an autoinhibitory function, whereby an internal NLS-like sequence competes for the importin-a binding site, reducing binding affinity for cargo proteins and helping to facilitate the release of the cargo within the nucleus [10,11] The removal of the autoinhibitory domain to create a truncated importin-a avoids possible competition for the binding site between this internal NLS and the CLIC4 NLS peptide The importin-a C-terminal domain (residues 70–529) consists of 10 ARM structural repeats that form two well-characterized cargo binding sites, referred to as the major and minor binding sites [1] These sites are located in the concave face of the protein near regions of invariant Trp and Asn arrays The major binding site spans ARM repeats 2, and (Fig 1A) In the major binding site, the positions of six NLS residues are labelled P1–P6, following the directionality of a bound NLS from the N-terminus to the C-terminus, which runs antiparallel to importin-a Our structure shows that the CLIC4 NLS peptide binds to the importin-a major binding site in an extended conformation consistent with a classical importin-a:NLS complex There is no clear interaction between the CLIC4 NLS peptide and the minor binding site of importin-a In the major binding site, electron density clearly defines the peptide residues 201VAKKYRN207, which have been included in the final model with Lys203 occupying the critical P2 binding position Our results reveal that Lys199 at the putative CLIC4 NLS N-terminus is disordered in the crystal and is therefore not necessary for peptide binding The core binding pockets P2–P5 are occupied by residues KKYR, a rather atypical NLS because of the presence of a bulky aromatic Tyr residue in the P4 binding position Surprisingly, the Tyr205 side-chain is favourably placed at P4, forming hydrogen bonds with the importin-a main chain An analysis of normalized B factors demonstrates a localized reduction in atomic flexibility experienced by importin-a residues as a result of the binding of the CLIC4 NLS peptide FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1663 Crystal structure of importin-a:CLIC4 NLS peptide A V Mynott et al The importin-a:CLIC4 NLS structure presented in this article adds to a growing body of knowledge on the structural mechanisms that govern the classical nuclear import model It also clarifies that the CLIC4 NLS can indeed bind directly to importin-a on condition that it can unfold into an extended conformation Results Structure of the importin-a:CLIC4 NLS peptide complex The structure of importin-a (70–529) bound to the CLIC4 NLS peptide (198VKVVAKKYRN207) was ˚ solved at 2.0 A resolution using synchrotron radiation (Table 1) The model of importin-a in the CLIC4 NLS peptide complex includes residues 72–496 and closely resembles the full-length importin-a structure that incorporates the N-terminal autoinhibitory domain ˚ (PDB:1IAL, rmsd of 0.20 A across 425 Ca atoms in residues 72–496) The major binding site spanning ARM repeats 2–4 has a similar conformation to the equivalent region in apo importin-a (70–529), with an ˚ rmsd of 0.16 A (46 Ca atoms) across the inner H3 helices, suggesting that there are minimal backbone conformational changes as a result of peptide binding Electron density corresponding to residues 201–207 (VAKKYRN) of the CLIC4 NLS peptide was unambiguously identified in the importin-a major binding site between ARM repeats 2–4 The Fo ) Fc map constructed by omitting the peptide from model phases is shown in Fig 1A The importin-a minor binding site contains no electron density that unambiguously corresponds to the CLIC4 NLS, and therefore no peptide was modelled at this site The CLIC4 NLS binds in an extended conformation that runs antiparallel to importin-a, analogous with other NLS cargo The average atomic B factors for importin-a in the structure are ˚ ˚ 32.1 A2 for main-chain atoms, 35.7 A2 for side-chain ˚ overall (3244 atoms) For the pepatoms and 33.8 A tide, B factors are slightly higher than those for ˚ ˚ importin-a: 36.9 A2 for main-chain atoms, 40.2 A2 for ˚ overall (62 total atoms) side-chain atoms and 38.7 A Electron density analysis Both the main-chain and side-chain atoms of the modelled CLIC4 peptide show a good fit to the electron density (Fig 1B) The peptide residues 202–207 correspond to the key binding positions P1–P6, with the critical P2 position occupied by Lys203 This means that the core basic motif, 203KKYR206, fills the central binding pockets P2–P5 in which the majority 1664 of peptide side-chain interactions take place with importin-a Therefore, the CLIC4 NLS satisfies the accepted consensus sequence for an optimal NLS, P2 K(K ⁄ R)·(K ⁄ R)P5 [12] The residue at P4, which has been shown to contribute the least, energetically, to peptide binding of the four main binding pockets [12], is unambiguously occupied by Tyr205 as defined by 2Fo ) Fc electron density The N-terminal peptide residues, 198VKV, are disordered in the crystal and are thus likely to be highly flexible It is particularly noteworthy that the basic residue, Lys199, defined as part of the putative CLIC4 NLS (KVVAKKYR) [2], does not contribute to peptide binding If the N-terminal flanking region increases CLIC4 NLS affinity for importin-a, it is unclear how it does so from our crystal structure The terminating carboxyl group of the peptide at Asn207 is well defined in the 2Fo ) Fc electron density map, despite making no interactions with importin-a The presence of a Tyr residue at P4 is a strong indication that the bound peptide corresponds to the CLIC4 NLS There is also weak and unaccounted for ˚ density approximately A from the aromatic plane of Tyr205 in a position that may correspond to a cation–p interaction The cation in this case is likely to be an Na+ ion from the crystallization buffer, with a low occupancy (< 50%) As a result of the weak nature of the Tyr205 cation–p bond, it seems unlikely that it will have a significant effect on the CLIC4 NLS peptide binding to importin-a We have also analysed the veracity of the CLIC4 NLS model built in the major binding site by inspectapo clic4nls À Fo data–data difference Fourier (see ing the Fo Materials and methods) This Fourier analysis reduces bias when interpreting the density of a peptide bound to importin-a and thus provides additional support for our structure The results are shown in Fig 1D As expected, the electron density is strong along the peptide main chain with well-defined carbonyl and amide backbone groups The one exception to this is the location of the amide group of Lys204 at P3, where there is a break in the main-chain density at the 2.8r map level The corresponding position in the apo structure has particularly strong density at this point, which may correspond to a water molecule Peptide sidechain density is also well defined in the F clic4nls ÀF apo o o map At P1, the density is weak, but resembles Ala202 The Lys residue at P2 (Lys203) is well defined despite the presence of a partially occupied water at this location in the apo structure Lys204 at P3 and Arg206 at P5 are also well defined Perhaps the most definitive characteristic of the F clic4nls ÀF apo difference Fourier o o is the strong and unambiguous electron density FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS A V Mynott et al Crystal structure of importin-a:CLIC4 NLS peptide Fig The importin-a:CLIC4 NLS peptide complex (A) The Fo ) Fc ‘omit’ electron density map over all atoms in importin-a Positive contours are shown at 2.8r in grey Density corresponding to the bound CLIC4 NLS peptide is clearly visible in the major binding site (B) Stereoimage of the CLIC4 NLS peptide and 2Fo ) Fc map The CLIC4 NLS peptide bound to the importin-a major binding site is shown as a stick representation Colour code for atoms: carbon, cyan; nitrogen, blue; oxygen, red Electron density is contoured at 1.5r in grey Binding ˚ positions P1–P6 and the N- and C-termini are labelled (C) Schematic representation of hydrogen bonds (broken lines, < 3.5 A) between importin-a and the CLIC4 NLS peptide, P1AKKYRNP6 Backbone carbonyl oxygens and amide nitrogens are shown as red and blue spheres, respectively Nitrogen and oxygen side-chain atoms are shown as blue and red squares, respectively (D) Stereoimage of the CLIC4 NLS bound to importin-a, showing the F clic4nls ÀF apo data–data difference Fourier map Grey contours represent positive difference density at o o ˚ 2.8r (E) Stereoimage of hydrogen-bond interactions (broken lines, < 3.5 A) The CLIC4 NLS peptide is shown as a ball and stick representation, where carbons are black, nitrogens are blue and oxygens are red Importin-a is shown in cartoon representation (cyan) with bonded residues shown as sticks corresponding to Tyr205 at P4 There is a strong positive difference density peak near the Tyr205 O–Cf bond at the 9.1r map level, the strongest density peak in the F clic4nls ÀF apo difference map The presence of o o Tyr205 is definitive evidence that the CLIC4 NLS peptide binds importin-a (70–529) CLIC4 NLS interactions with importin-a The CLIC4 NLS forms an extensive network of interactions with importin-a through both main-chain and side-chain atoms, similar to other importin-a structures with a bound monopartite NLS [13–15] Hydrogen FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1665 Crystal structure of importin-a:CLIC4 NLS peptide A V Mynott et al Fig Analysis of the importin-a:CLIC4 NLS peptide complex (A–C) Importin-a is coloured by the normalized B factor score, B Àapo , over a z blue–magenta colour spectrum ()3r to +3r) (A) The bound CLIC4 NLS is shown on the molecular surface of importin-a in the major binding site (B) The importin-a Ca backbone is shown as a cartoon tube representation in the same orientation as in (A) Important residues are shown as a stick representation (C) Full-length images of importin-a coloured by the B Àapo score Residues in grey have not been included z in the calculation of B Àapo (D) The CLIC4 crystal structure (PDB:2AHE) is shown as a cartoon representation The N-terminal thioredoxin z domain (blue) and C-terminal a-helical domain (green) are coloured separately The CLIC4 NLS residues are highlighted in cyan Inset: The NLS is shown as a stick representation (carbons, cyan; oxygens, red; nitrogens, blue) Hydrogen bonds are represented by broken lines (E) A multiple sequence alignment of the CLIC4 NLS motif in human CLICs Conserved residues are red, nonconserved residues are black and perfect conservation is highlighted with red fill The sequence of CLIC3 is added for comparison Binding positions P1–P6 are shown in an alignment corresponding to our importin-a:CLIC4 NLS peptide complex Sequence alignment was performed using CLUSTALW [44] and ESpript [45] bonding by the main chain of the CLIC4 NLS peptide involves importin-a side chains in the conserved WxxxN motifs of ARM repeats 2-4, which include residues Trp142, Trp184, Asn146, Asn188 and Asn235 (Fig 1C) In the CLIC4 NLS peptide, this corresponds to hydrogen-bonded carbonyl and amide groups from every second residue in the major binding site: P1 (Ala202), P3 (Lys204) and P5 (Arg206) The peptide side chains in binding positions P2 (Lys203), P4 1666 (Tyr205) and P5 (Arg206) form hydrogen bonds to the importin-a main chain and side chains In addition, Lys203 forms a critical salt bridge with impaAsp192 ˚ (‘impa’ denotes importin-a; bond length, 2.80 A; Nf ) Od) at P2, the most energetically significant interaction involved in importin-a recognition of NLSs [16,17] Other basic residues in the peptide, Lys204 and Arg206, fill negatively charged pockets at P3 and P5 FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS A V Mynott et al Crystal structure of importin-a:CLIC4 NLS peptide Table Data collection and refinement statistics Data collection Source (k) Australian Synchrotron ˚ (0.95 A) ADSC Q210 P212121 78.6, 89.6, 100.1 2.0 (2.11–2.00) 318 168 (27 080) 46 758 (5520) 96.7 (80.3) 12.2 (1.8) 9.6 (77.6) 30.4 Detector Space group ˚ Unit cell dimensions (A): a, b, c ˚ Resolution (A)a Observations Unique reflections Completeness (%)a Mean I ⁄ Rmerge (%)a,b ˚ Wilson B value (A2) Refinement and structure R factor (%)c 19.6 Rfree (%) 23.6 Scaling R factor (Riso)d 16.3 ˚ Number of nonhydrogen atoms (mean B value, A2) Importin-a 3238 (35.2) Peptide 62 (40.4) Water 356 (45.5) Ramachandran plot (%)e Favoured region 98.6 Allowed region 1.4 Disallowed a Outer shell statistics are shown in parentheses b Rmerge = RhklRi|Ii ) | ⁄ RhklRiI c R factor = Rhkl||Fobs| ) |Fcalc|| ⁄ Rhkl|Fobs| d Riso P P ¼ hkl jF clic4nls ÀF apo j= hkl jF apo j e Calculated by MolProbity [18] o o o without forming salt bridges At P3, there is a pocket formed between impaTrp184 and impaTrp231 which favourably accepts the extended and positively charged ˚ Lys204 side chain, with impaGlu266 (5.8 A, Nf ) Oe impa ˚ , Nf ) Od distance) distance) and Asp270 (4.5 A positioned at the end of the binding pocket Similarly, the P5 pocket formed between impaTrp142 and impa Trp184 favourably accepts Arg206, with impaGlu180 ˚ (4.9 A, Nf1 ) Oe1 distance) positioned at the end of the binding pocket In total, the CLIC4 NLS main chain and side chains make 174 atom-to-atom van der Waals’ contacts with importin-a and 13 hydrogen bonds, including one salt bridge at P2 In addition, there are 10 hydrogen bonds formed between the peptide and water molecules, three of which involve the terminating carboxyl group The van der Waals’ contact area between the CLIC4 NLS peptide and importin-a has been calculated for each peptide residue by integrating over contact areas using MolProbity [18,19] Main-chain contributions to the contact area were found to be approximately equal ˚ (2–5 A2) Side-chain contributions vary to a greater extent, reflecting differences in the binding pockets The Lys and Arg residues in binding positions P2 and P5 have the largest side-chain van der Waals’ contact ˚ areas, both corresponding to 15.9 A2 This is closely followed by the Tyr residue at P4, with a contact area ˚ of 13.5 A2 and the Lys residue at P3 with a contact ˚ area of 10.2 A2 Residues outside the central binding area have significantly lower values These results are summarized in Table The solvent-accessible surface area on importin-a ˚ buried by the peptide is 513.1 A2, with the largest contribution of buried surface area from the Trp array ˚ ˚ that includes Trp142 (51.3 A2), Trp184 (70.2 A2) and ˚ 2) The surface area buried on the pepTrp231 (63.7 A ˚ tide is 744.7 A2, which corresponds to 59.2% of the total peptide surface area The real space correlation coefficient (RSCC) for each residue has also been calculated by comparing the importin-a:CLIC4 NLS experimental electron density with density calculated from the model The CLIC4 NLS main chain fits the density well, with an average RSCC of 0.96 Side chains have greater RSCC variability, with an average of 0.89 over all residues and 0.94 for those in P2-P5 (Table 2) The CLIC4 NLS Tyr residue By solving the structure of the importin-a:CLIC4 NLS complex, we have shown that the major binding pocket P4 is unambiguously occupied by a Tyr residue: the first importin-a:NLS structure that has an aromatic residue present in the core binding region Tyr205 adopts a common rotamer with a score of 82.9% (v1 $ 180, v2 $ 80), calculated by comparing the side chain with a high-quality reference dataset using MolProbity [18] Interestingly, it appears that this unique NLS residue is not only accommodated in the P4 site, but makes significant interactions with importin-a This primarily occurs by the formation of a hydrogen bond between the side-chain hydroxyl group of Tyr205 and the importin-a main chain at the C-terminus of the ARM1 H3 helix The hydrogen bond is possibly shared between the carbonyl oxygen of impaLeu104 ˚ (bonding distance from oxygen to oxygen, 2.83 A) and impa Arg106 (oxygen to oxygen, the carbonyl oxygen of ˚ 2.64 A) Although the amide nitrogen of impaArg106 ˚ (N–O, 3.16 A) is of hydrogen-bonding distance, the geometry for this interaction is not favourable Analysis of importin-a:CLIC4 NLS contacts using MolProbity suggests that the Tyr205–Arg106 (O–O) hydrogen bond is the most favourable with optimal geometry (refer to Fig S1) The bulky side chain of Tyr205 also makes extensive hydrophobic interactions with surrounding importin-a FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1667 Crystal structure of importin-a:CLIC4 NLS peptide A V Mynott et al Table Characteristics of the importin-a:CLIC4 NLS peptide interaction ˚ Van der Waals’ contact area (A2)b Interactionsa NLS residue Main chain Side chain Main chain Side chain Total Buried surface ˚ area (A2) RSCCc Val201 Ala202 P2 Lys203 P3 Lys204 P4 Tyr205 P5 Arg206 P6 Asn207 Totals 0⁄1⁄0 1⁄8⁄0 0⁄6⁄1 3⁄5⁄0 0⁄8⁄0 3⁄9⁄0 0⁄6⁄3 ⁄ 42 ⁄ 0⁄0⁄0 0⁄6⁄0 ⁄ 31 ⁄ 1 ⁄ 20 ⁄ ⁄ 30 ⁄ 1 ⁄ 45 ⁄ 0⁄0⁄0 ⁄ 132 ⁄ 0.4 3.5 2.2 3.3 5.6 2.8 20.8 5.3 15.9 8.0 13.5 15.9 58.6 0.4 8.8 18.9 10.2 16.8 21.5 2.8 79.4 26.7 89.4 145.3 128.4 133.8 174.8 46.3 744.7 0.89 ⁄ 0.74 0.91 ⁄ 0.81 0.96 ⁄ 0.97 0.98 ⁄ 0.92 0.96 ⁄ 0.93 0.98 ⁄ 0.94 0.96 ⁄ 0.79 0.96 ⁄ 0.89 P1 a Shown as importin-a hydrogen bonds ⁄ van der Waals’ contacts ⁄ ordered solvent hydrogen bonds b Calculated using MolProbity by integrating ˚ contact dots with 16 dots ⁄ A2 [18] c The real space correlation coefficient (RSCC) is listed as main chain ⁄ side chain residues, whereby the phenol ring fits into a hydrophobic pocket formed by the loop connecting ARM repeats and (Leu104–Pro111) Indicative of the tightness of the fit, there are a large number (30) of atom-to-atom van der Waals’ contacts between the Tyr205 side chain and importin-a (Table 2) The numbers of contacts are comparable with those of the basic residues at P2 (Lys203: 31 contacts), P3 (Lys204: 20 contacts) and P5 (Arg206: 45 contacts) Normalized B factor analysis In order to analyse changes in the conformational dynamics of importin-a binding site residues, as a result of the presence of a bound CLIC4 NLS peptide, the relative B factor score, BÀapo , was calculated This z score represents a change in flexibility of each residue in apo importin-a and the corresponding residue in the importin-a:CLIC4 NLS complex The BÀapo score was z determined by comparing normalized B factors from the importin-a:CLIC4 NLS structure with those from apo importin-a (see Materials and methods) A negative score represents a decrease in flexibility, a positive score represents an increase in flexibility and a score of zero represents no change Residues near the importina C-terminus (430–496) were considered to be outliers (Z > 4) and are thus excluded from the analysis The BÀapo scores have a zero mean and standard deviation z of unity (Fig S1) The BÀapo score is colour mapped z onto a molecular surface representation of the importin-a molecule in Fig Here, the scores were averaged over main-chain (BÀapo mc ) atoms and side-chain z (BÀapo sc ) atoms for each residue z We note that the major binding site corresponds to a cluster of negative BÀapo scores, demonstrating a z reduced flexibility on binding the CLIC4 NLS peptide, whereas the minor binding site is unchanged This is 1668 particularly significant over the Trp and Asn arrays, which includes residues that interact directly with the CLIC4 NLS peptide backbone: Asn146 ()4.8sc); Asn188 ()2.4sc); Asn235 ()2.0sc); Trp142 ()2.2sc); Trp184 ()3.9sc) In addition, Ser149 ()3.3sc) is notably constrained by the CLIC4 NLS in a single conformation compared with dual rotamer conformations in the apo structure The presence of the Lys203 side chain at P2 has marginal effects on the hydrogen-bonding partners Thr155 ()0.6res) and Asp192 ()0.8res), but a significant effect on the third hydrogen-bonding partner, the carbonyl group of Gly150 ()3.1res) This can be explained by the presence of a water molecule in the apo structure which is predicted to make hydrogen bonds with Thr155 and Asp192, but not Gly150 We also note that the Lys Nf–Gly150 bond has the added effect of reducing flexibility in the connecting loop between ARM repeats and (Ser149–Ser152) At the P4 binding site, we see that Tyr205 has a stabilizing effect on residues in the ARM1–ARM2 connecting loop The hydrogen bond formed between Tyr205 and the carbonyl oxygen of impaArg106 ()2.8res) decreases significantly the BÀapo score for the z residue This is also seen for the other Tyr205-shared hydrogen-bonding partner, impaLeu104 ()2.3res) Van der Waals’ contacts involving Tyr205 appear to stabilize both impaPro110 ()1.2res) and impaSer105 ()3.3res) The CLIC4 NLS tightens residue mobility across the ARM2–ARM3 connecting loop from Leu103 to Ile112 Arg206 at P5 forms a hydrogen bond with impaGln181 which has a corresponding decrease in its BÀapo score of z )1.9sc This B factor analysis does not reflect significant changes in residue flexibility as a result of long-range electrostatic interactions between CLIC4 NLS side chains at P3 (Lys204) or P5 (Arg206) and acidic residues in importin-a (Glu180, Glu266, Asp270) FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS A V Mynott et al Discussion The importin-a:CLIC4 NLS peptide complex ˚ In this article, we have presented the 2.0 A resolution X-ray crystal structure of the CLIC4 NLS peptide (198VKVVAKKYRN207) bound to importin-a (70– 529) The monopartite NLS peptide binds in the major binding groove of importin-a in an extended conformation consistent with previously solved structures [1] In the case of CLIC4, this extended NLS conformation differs greatly from its helical conformation as seen in the soluble CLIC4 structure (Fig 2D) This important feature means that the putative CLIC4 NLS needs to undergo a structural transition if it is to be a biologically active NLS It also suggests that there is tighter control over CLIC4 translocation to the nucleus in comparison with other nuclear-destined proteins that not require their NLS to undertake a structural rearrangement The F clic4nls ÀF apo data–data difference Fourier was o o inspected to ensure that electron density in the importin-a major binding site is not confused with residual density from the apo importin-a structure The results of this difference Fourier show that all peptide side chains in P1–P6 positions have well-defined F clic4nls ÀF apo density, supporting the assignment of o o CLIC4 NLS residues in these binding positions This analysis is therefore a recommended method of structural examination in other importin-a:NLS complexes to ensure the correct identification of the bound peptide Side-chain interactions involving CLIC4 NLS Lys residues at P2 and P3 are equivalent to those observed in the importin-a:simian virus 40 (SV40) large T-antigen (TAg) NLS complex [13] This includes the key electrostatic interaction at P2 involving Lys203 and impa Asp192 In classical NLSs, the Lys at P2 is known to provide the most substantial energetic contribution to peptide binding [12] and is critical for nuclear import, which can be eliminated through mutagenesis of this single amino acid [16] Although the work presented by Suh et al [2] obliterated the putative CLIC4 NLS motif (199KVVAKKYR206 to 199TVVAITYG206), rendering CLIC4 unable to translocate to the nucleus, a single point mutation at Lys203 should be sufficient to prevent nuclear translocation A prominent difference between the CLIC4 NLS and classical NLSs is the presence of a bulky aromatic residue within the core NLS motif Our structure shows how a Tyr residue can be accommodated in the major binding site at P4, and its presence is confirmed by strong, unambiguous density in the F clic4nls ÀF apo o o Crystal structure of importin-a:CLIC4 NLS peptide difference map We note that a structure of the phospholipid scramblase NLS bound to importin-a has been reported, in which a Trp residue is located three residues downstream from P4 [20] The Tyr residue is found to interact with importin-a through both hydrophobic and hydrogen-bonding interactions Although it is an unusual NLS residue containing a bulky phenol side group, it neatly fits at its location with the hydroxyl group reaching the importin-a main chain near the C-terminus of the ARM1 H3 helix Most of the hydrophobic contacts with importin-a residues lining the P4 pocket are equivalent to those made by the aliphatic portion of an Arg residue in the TAg NLS, whereas the hydrogen bonds shared between the Tyr hydroxyl group and carbonyl groups from impaLeu104 and impaArg106 are reproduced in the importin-a:TAg NLS P4 binding position involving the Nf atom of TAgArg130 Because of the proximity of the Tyr205 hydroxyl group to importin-a, CLIC4 NLS peptide binding should be prevented by Tyr phosphorylation Therefore, the phosphorylation state of CLIC4 could foreseeably act as a switch between an ‘active’ and ‘inactive’ CLIC4 NLS Structural studies have shown that the importin-a P4 binding position tolerates a wide range of residues, including Arg from the TAg NLS (PDB:1EJL [13]), Lys in the nucleoplasmin NLS (PDB:1EE5 [21]) and androgen receptor NLS (PDB:3BTR [14]), Val in the c-myc NLS (PDB:1EE4 [21]), Leu in the retinoblastoma NLS or Ser in the N1N2 NLS (1PJM and 1PJN [22]) and Ile in the influenza A PB2 subunit NLS (PDB:2JDQ [23]) However, an oriented peptide library screening study demonstrated that Tyr has a higher specificity for the P4 binding position compared with these hydrophobic residues (Val, Leu and Ile) [24] Interestingly, the contribution to NLS binding energy of the residue at P4 is similar whether it is occupied by a positively charged Arg side chain or a hydrophobic Val side chain [12] This is despite the extra hydrogen bonding formed by Arg at P4 and a possible helix dipole interaction with H3 helices of ARM repeats and Although the hydrogen bonds formed by Tyr205 may suggest that a Tyr at P4 is more favourable than a Phe or other bulky residue, it is likely to have a similar energetic contribution to peptide binding as Val or Arg The CLIC4 NLS contains an Arg residue (Arg206) in binding position P5 which adopts a common and extended rotamer conformation The P5 position is defined by importin-a residues Trp142 and Trp184, which both adopt a rare rotamer conformation, facilitating the formation of this hydrophobic pocket The site is normally occupied by a NLS Lys residue in FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1669 Crystal structure of importin-a:CLIC4 NLS peptide A V Mynott et al importin-a:NLS structures, where the aliphatic portion of the side chain fits into the hydrophobic alcove and allows the charged head group access to the solvent on the other side The extended side chain of Arg206 allows for the same favourable interactions as a Lys residue, including a hydrogen bond to impaGln181 and the formation of a network of solvent interactions The positively charged guanidinium group of Arg206 is also compensated by the nearby acidic residue impa Glu180, with an interaction distance (Nf1 ) Oe1) of ˚ 4.9 A Experiments showing that CLIC4 translocates to the nucleus are not clear on the exact mechanism by which translocation occurs Although mutagenesis of the putative CLIC4 NLS implies an interaction with importin-a, immunoprecipitation experiments have shown that CLIC4 also associates with nuclear transport factor-2 and Ran [2] The nuclear transport factor-2 pathway is normally used for the nuclear import of RanGDP [28], but nuclear transport factor-2 can also interact with cargo complexes [29] The nuclear transport of CLIC4 may therefore reveal new general paradigms for nucleocytoplasmic transport processes Conservation of the CLIC NLS The CLIC4 NLS 199KVVAKKYR206 is highly conserved in vertebrate species and across CLICs 1–6, apart from CLIC3 (Fig 2E) The N-terminal KVV motif in the CLIC NLS is mostly conserved; however, its importance to aiding the recognition by importin-a is likely to be small, as we observed no interaction between these residues and importin-a in our structure This is particularly noteworthy considering the presence of Lys199 in this flanking region We therefore suggest that the CLIC4 NLS motif, currently reported as 199KVVAKKYR206, could be abbreviated to include only those residues that bind directly to importin-a, 202AKKYRN207 The KKYR motif, which occupies the core binding region P2–P5, controls importin-a recognition of the NLS and is conserved in all human CLICs, except CLIC3 The significance of the motif is clear, with 92% of the hydrogen bonds formed between importina and the CLIC4 NLS occurring in this region It is interesting to note that the KKYR motif is ignored by NLS predicting tools, such as PredictNLS [25] and NLStradamus [26] This suggests that the criteria for predicting an NLS need to be expanded to account for residue variability, particularly in the P4 binding position Future studies may seek to elucidate the importance of the KKYR motif in governing the nuclear import for other CLICs, and we note that CLIC1 has been shown to localize to the nucleus with an unexplained import pathway [27] Import pathways The importin-a:CLIC4 NLS complex presented here supports the hypothesis that CLIC4 can enter the nucleus via an importin-a-mediated nuclear import pathway However, the recognition of full-length CLIC4 by importin-a is strictly dependent on its ability to undergo a change in conformation that exposes a linear NLS ready for binding 1670 Triggers for structural change Typically, NLSs are located in solvent-exposed regions of unstructured domains, such as flexible ends or loop regions The CLIC4 NLS motif, as seen in the soluble CLIC4 crystal structure, is unusual for an NLS as it is neither unfolded nor situated at domain termini (Fig 2D) As we have shown that the CLIC4 NLS binds to importin-a in an extended conformation, this confirms a previous hypothesis [4] that structural changes in CLIC4 are required to expose the NLS region The NLS in CLIC4 forms a structurally stable helical conformation at the C-terminus of helix The key KKYR binding motif in the NLS is positioned so that Arg206 terminates the helix and the two Lys residues are solvent exposed A number of side-chain hydrogen-bonding interactions involve helix and the C-terminal tail Given the metamorphic nature of the CLIC module, as demonstrated by the structural transition of monomeric CLIC1 to a dimeric form [30], we anticipate that there is a structural rearrangement that can unfold the CLIC4 NLS A recent study has observed a post-translational modification to CLIC4 which could represent the required trigger for structural change [3] This modification is the S-nitrosylation of a Cys residue located between helix and helix 9, near the CLIC4 NLS site It has been shown that Cys234 is S-nitrosylated when exposed to a nitric oxide agent, S-nitrosocysteine [31], despite the thiol side chain being buried in an interdomain interface Consistent with this hypothesis is a correlation between nitric oxide donors and the rate of nuclear translocation of CLIC4 [32], as well as an increased association between CLIC4 and importin-a after nitrosylation [3] Using the high-resolution crystal structure of human thioredoxin (PDB:2HXK), which contains a buried S-nitrosocysteine (Cys62), the coordinates of the SNO group were transferred to Cys234 in CLIC4 (refer to Fig S1) Both Cys residues adopt the most common FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS A V Mynott et al rotamer assisting nitric oxide transfer In this model, a strong steric clash is expected between the S-nitrosylated Cys234 and His196 in helix 6, which can be allevi˚ ated by a shift of at least A in the interdomain interface Accordingly, experiments have shown that the S-nitrosylation of CLIC4 induces a structural change [3] We note that an S-nitrosylation trigger leading to conformational change has been observed previously in blackfin tuna myoglobin, where the S-nitrosylation of a Cys residue causes a structural domain shift [33] Whether the S-nitrosylation of Cys234 results in a similar structural rearrangement of CLIC4, and provides a trigger for NLS conformational change, requires further investigation The possibility of trigger events to expose NLSs offers an interesting criterion for cargo selection by nuclear import receptors Typically, NLSs are identifiable as a result of their high content of basic residues, which allows them to bind tightly to an acidic binding site in importin-a However, it is clear that the prerequisite for NLS binding, based on sequence alone, is very limited because a core region of just four residues is necessary for importin-a peptide recognition Furthermore, there is a certain degree of tolerance to nonbasic residues within the importin-a binding region, particularly at P4, where we find the Tyr residue in CLIC4 NLS In order to select specific cargo proteins destined for the nucleus, it is likely that importin-a screens additional criteria, such as the flexibility of the NLS and its solvent exposure within a folded protein This form of regulation is governed more by the cargo protein itself, rather than importin-a, and, as such, is a specific control mechanism that has not been thoroughly considered among the many levels of import regulation For a protein such as CLIC4, in which nuclear translocation is linked to cellular events, it is understandable that a higher level of regulation is needed relative to proteins that regularly shuttle across the nuclear membrane The folded state of the CLIC4 NLS may provide the necessary barrier to binding importin-a, so that an environmental trigger is required before the NLS unfolds into an extended and exposed sequence that is recognized by the import receptor Materials and methods Protein expression and purification The generation of the mouse importin-a (70–529) expression construct has been described previously [13] The construct consists of residues 70–529 from Mus musculus importin-a isoform a2, inserted into the pET-30a N-terminal His-tag expression vector (Novagen, Madison, WI, USA) The plas- Crystal structure of importin-a:CLIC4 NLS peptide mid was transformed into BL21 (DE3) cells and grown at 37 °C overnight in Luria–Bertani medium containing 30 lgỈmL)1 of kanamycin Flasks of 400 mL of Luria– Bertani medium (30 lgỈmL)1 kanamycin) were then inoculated with 10 mL of the overnight culture The cells were grown at 37 °C until an absorbance at 600 nm of 1.0 cm)1 was reached, at which point the culture was induced with mm isopropyl thio-b-d-galactoside The temperature was then lowered to 30 °C and expression continued for approximately h Cells were harvested by centrifugation at 11 000 g for and resuspended in 35 mL NaCl ⁄ Pi with mm dithiothreitol At this stage, protease inhibitors were also added (Complete Protease Inhibitor Cocktail; Roche Applied Science, Penzberg, Germany) The cells were lysed using a French pressure cell and the resulting cell debris was centrifuged at 39 000 g for 45 at °C The supernatant was added to a 3-mL solution of either nickel nitrilotriacetic acid agarose resin (Qiagen, Valencia, CA, USA) or Profinity immobilized metal affinity chromatography (IMAC) resin (Bio-Rad, Hercules, CA, USA), and was mixed under gentle rotation for h The resin was then loaded into an Econopak gravity flow column (Bio-Rad) and unbound protein was eluted and discarded The resin was washed with 200 mL of 20 mm Hepes, pH 7.0, 500 mm NaCl, mm MgCl2, 0.1 mm dithiothreitol and up to 20 mm imidazole Bound protein was then step eluted with 10 mL of a buffer containing 20 mm Hepes, pH 7.0, 500 mm NaCl, 0.1 mm dithiothreitol and 150 mm imidazole In addition to step elution of importin-a (70–529) after binding to IMAC resin, an imidazole gradient was employed in some instances for greater control over protein elution and higher purity A HisTrapÔ FF column (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK) with a volume of mL and prepacked with Ni-SepharoseÔ Fast Flow resin was first pre-equilibrated with a buffer consisting of 20 mm Tris, pH 8.0, and 500 mm NaCl before binding, and then washed with 20 mm Tris, pH 8.0, 500 mm NaCl, mm MgCl2 and 20 mm imidazole The protein was eluted over an imidazole gradient from 20 to 250 mm at a flow rate of 0.5 mLỈmin)1 over a volume of 40 mL Fractions with the majority of protein, as assessed by UV absorption at 280 nm (A280), were then pooled and dialysed into 20 mm Tris, pH 8.0, 100 mm NaCl and mm dithiothreitol The purification protocol described here follows the methods reported in the literature [13,22] For ion exchange chromatography, protein was loaded onto a HiloadÔ 26 ⁄ 10 Q-SepharoseÔ anion exchange column (GE Healthcare) immediately after imidazole gradient IMAC purification Importin-a was allowed to bind to the N+(CH3)3 charged groups of the Q-SepharoseÔ HP resin in Buffer A (50 mm NaCl, 20 mm Tris, pH 8.0, mm dithiothreitol), before being eluted over a salt gradient from 50 mm to m at a flow rate of mLỈmin)1 (total volume FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1671 Crystal structure of importin-a:CLIC4 NLS peptide A V Mynott et al of 400 mL) Eluted fractions with the highest concentration of protein were pooled and concentrated using an Amicon YM-10 Centriprep centrifugal concentrator (Amicon, Billerica, MA, USA) in a Sorvall SH-3000 swinging bucket rotor (Sorvall, Waltham, MA, USA) at 1800 g The concentrated protein from ion exchange chromatography was loaded on a Superdex 200 10 ⁄ 300 GL (GE Healthcare) analytical size exclusion column and eluted with 20 mm Tris, pH 8.0, 100 mm NaCl and mm dithiothreitol Fractions corresponding to importin-a (70–529) were pooled and concentrated to approximately 18 mgỈmL)1, flash frozen in liquid nitrogen and stored at )80 °C until use NLS peptide synthesis The CLIC4 NLS peptide was synthesized (Sigma-Genosys, Sydney, Australia) and used in the crystallization of importin-a:CLIC4 peptide complexes Although the proposed CLIC4 NLS motif includes residues 199–206 (199KVVAKKYR206) [2], the CLIC4 NLS synthetic peptide used for crystallization includes one-residue extensions at both termini (NH2-198VKVVAKKYRN207-COOH) The purity was assessed by analytical RP-HPLC to be better than 95% and the peptide has a molecular mass of 1204.5 Da and pI of 10.9 Lyophilized peptides were first dissolved in water or a suitable assay buffer before use The stock concentration of each peptide sample varied between and mgỈmL)1 for different crystallization trials Crystallization and data collection The importin-a (70–529):CLIC4 NLS crystals were obtained by co-crystallization using hanging drop vapour diffusion The reservoir consisted of 500 lL of 0.7 m sodium citrate, 10 mm dithiothreitol and 70 mm Hepes at pH 7.4 Drops consisted of 0.75 lL of importin-a (70-529) at 18 mgỈmL)1, 0.75 lL of CLIC4 NLS peptide at mgỈmL)1 and lL of reservoir These conditions resulted in a 7.6 times molar excess of the CLIC4 NLS peptide over importin-a (70–529) There was no incubation of peptide and protein other than the immediate mixing in the crystallization drop Crystals grew to maturity in approximately weeks at 20 °C Crystals were gradually transferred into a cryoprotectant solution consisting of the reservoir solution supplemented with 20% glycerol The importin-a:CLIC4 NLS crystal was flash cooled in liquid nitrogen and stored in a cryogenic dry shipping dewar It was then mounted at 100 K in a nitrogen cryostream on beamline PX-1 (3BM-1B) at the Australian Synchrotron [34], and data collection was performed using an ADSC Quantum 210 (Q210) detector A number of initial images were screened at orthogonal u angles to gauge crystal quality before autoindexing and determining the optimum rotation range using strategy from within mosflm [35] Crystals were found to have the symmetry of the orthorhombic space group P212121 with 1672 ˚ ˚ unit cell dimensions of approximately a = 79 A, b = 90 A ˚ (see Table 1) The final diffraction dataset and c = 100 A was obtained over 180 images with a 1° oscillation angle (u) and 5-s exposure time per image The crystal to detector distance was set at 220 mm and the beam width was set to 200 lm · 200 lm Structure determination and refinement After datasets had been initially processed and integrated in mosflm, the reflection data file was then passed through a suite of programs in the ccp4 crystallography package [36] Scaling of intensities and inspection of data quality were performed in scala In particular, the resolution cut-off was determined by analysing the signal to noise parameter, , as it varies against resolution The resolution corresponding to an value of 2.0 was used as an approximate limit to the high-resolution bin, as described in the literature [37] The fraction of data used for the Rfree calculation was set to 5% Phases were determined using molecular replacement with the maximum likelihood search function in phaser [38] A model of importin-a was built using the full-length importina structure (PDB:1IAL [11]) as a search probe, with the solvent and NLS peptide model removed, and atomic B factors reset to an appropriate value as suggested from the Wilson plot An initial solvent model was built using arp ⁄ warp [39], which was then manually checked and validated using the visualization program coot [40] Water molecules that were not well ordered, as well as water molecules placed in or near the importin-a binding site, were removed Iterative refinement of the model was performed with multiple passes of refmac5 maximum likelihood refinement [41] and coot manual refinement Electron density for a bound peptide was generally much clearer in the major binding site compared with the minor binding site Data reduction and refinement statistics are summarized in Table Electron density analysis Fourier analysis was used to compare the importina:CLIC4 NLS peptide complex (observed structure factors F pep ) with an apo structure of importin-a (70–529) o (observed structure factors F apo ) This structure is described o in ref [42] The structure factors from each dataset were scaled using the ccp4 program scaleit [36] This calculates a scaling R factor of 16.3%, supporting isomorphism between the two crystals We refer to the Fourier difference of these structure factors as a data–data difference Fourier, F pep ÀF apo , which is calculated as: o o À pep   apo Á apo F ¼ Fo  À Fo  Á eiac where the phases (aapo ) are obtained from the apo imporc tin-a structure FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS A V Mynott et al Crystal structure of importin-a:CLIC4 NLS peptide B factor analysis In order to analyse changes in the conformational dynamics of importin-a residues, a comparison was made between the atomic B factors of apo importin-a and the importina:CLIC4 NLS complex These two crystals are isomorphous: they were both solved in the space group P212121, with similar lattice dimensions (CLIC4 NLS crystal: ˚ ˚ ˚ a = 78.6 A, b = 89.6 A, c = 100.1 A; apo crystal: ˚ ˚ ˚ a = 79.0 A, b = 89.8 A, c = 100.3 A), and the scaling R factor is relatively low (16.3%) We have calculated a normalized B factor score using a method described previously [43] The B factors of atoms in structure j are first normalized according to the following equation: Bi À l j Bz ¼ rj where Bz is the normalized B factor, Bi is the B factor of atom i, lj is the mean B factor (excluding water molecules and peptide atoms) and rj is the standard deviation of B factors The normalized B factors have a zero mean and unit variance All atoms that satisfy Bz ‡ are treated as outliers and discarded After discounting outliers, Bz values were recalculated The normalized B factors from apo importin-a were then subtracted from those in the importin-a:CLIC4 NLS structure, and the difference was again normalized using the equation above The final values are referred to as the BÀapo score, and were calculated for all nonhydrogen z 10 atoms in the importin-a:CLIC4 NLS structure Acknowledgements Data contributing to this research were obtained on the PX-1 beamline at the Australian Synchrotron, Vic., Australia 11 12 References Marfori M, Mynott A, Ellis JJ, Mehdi AM, Saunders ´ NFW, Curmi PM, Forwood JK, Boden M & Kobe B(2010) Molecular basis for specificity of nuclear import and prediction of nuclear localization Biochim Biophys Acta – Mol Cell Res doi:10.1016/j.bbamcr 2010.10.013 Suh KS, Mutoh M, Nagashima K, Fernandez-Salas E, Edwards LE, Hayes DD, Crutchley JM, Marin KG, Dumont RA, Levy JM et al (2004) The organellular chloride channel protein CLIC4 ⁄ mtCLIC translocates to the nucleus in response to 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Dunker AK & Zhu G (2003) Improved amino acid flexibility parameters Protein Sci 12, 1060–1072 44 Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673–4680 45 Gouet P, Courcelle E, Stuart DI & Metoz F (1999) ESPript: analysis of multiple sequence alignments in PostScript Bioinformatics 15, 305–308 Crystal structure of importin-a:CLIC4 NLS peptide This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors Supporting Information The following supplementary material is available: Fig S1 Analysis of the importin-a:CLIC4 NLS complex FEBS Journal 278 (2011) 1662–1675 ª 2011 The Authors Journal compilation ª 2011 FEBS 1675 ... A2 for main-chain atoms, 35.7 A2 for side-chain ˚ overall (3 244 atoms) For the pepatoms and 33.8 A tide, B factors are slightly higher than those for ˚ ˚ importin -a: 36.9 A2 for main-chain atoms,... atoms, 40 .2 A2 for ˚ overall (62 total atoms) side-chain atoms and 38.7 A Electron density analysis Both the main-chain and side-chain atoms of the modelled CLIC4 peptide show a good fit to the electron... fit, there are a large number (30) of atom -to- atom van der Waals’ contacts between the Tyr205 side chain and importin -a (Table 2) The numbers of contacts are comparable with those of the basic residues