Basis of recognition between the NarJ chaperone and the N-terminus of the NarG subunit from Escherichia coli nitrate reductase Silva Zakian 1 , Daniel Lafitte 2 , Alexandra Vergnes 1 , Cyril Pimentel 3 , Corinne Sebban-Kreuzer 3 , Rene ´ Toci 1 , Jean-Baptiste Claude 4 , Franc¸oise Guerlesquin 3 and Axel Magalon 1 1 Laboratoire de Chimie Bacte ´ rienne, Institut de Microbiologie de la Me ´ diterrane ´ e, Centre National de la Recherche Scientifique, Marseille, France 2 MaP site Timone, UMR INSERM 911, Universite ´ d’Aix-Marseille II, France 3 Interactions et Modulateurs de Re ´ ponses, Institut de Microbiologie de la Me ´ diterrane ´ e, Centre National de la Recherche Scientifique, Marseille, France 4 Information Ge ´ nomique et Structurale, Marseille, France Introduction A new family of molecular chaperones, conserved in most prokaryotes, performs essential roles in the biogenesis of both exported and nonexported metallo- proteins [1,2]. They share a common fold composed entirely of a-helices and several flexible regions [1,2]. A particular feature of these chaperones is their ability to interact with twin-arginine signal sequences of exported metalloenzymes or N-terminal sequences of nonexported ones [2,3]. The mechanisms governing such interactions are of paramount importance in the context of metalloprotein biogenesis. These interactions are well illustrated by the non- exported membrane-bound nitrate reductase complex (NarGHI) of Escherichia coli, harbouring no fewer than eight metal centres in three distinct subunits [4–6], and the NarJ chaperone. Dynamic interactions Keywords chaperone; metalloproteins; nitrate reductase; NMR; translocation Correspondence A. Magalon, Laboratoire de Chimie Bacte ´ rienne, Institut de Microbiologie de la Me ´ diterrane ´ e, Centre National de la Recherche Scientifique, 31, chemin Joseph Aiguier 13402 Marseille Cedex 09, France Fax: +33 491 718 914 Tel: +33 491 164 668 E-mail: magalon@ifr88.cnrs-mrs.fr (Received 8 December 2009, revised 25 January 2010, accepted 4 February 2010) doi:10.1111/j.1742-4658.2010.07611.x A novel class of molecular chaperones co-ordinates the assembly and targeting of complex metalloproteins by binding to an amino-terminal peptide of the cognate substrate. We have previously shown that the NarJ chaperone interacts with the N-terminus of the NarG subunit coming from the nitrate reductase complex, NarGHI. In the present study, NMR structural analysis revealed that the NarG(1–15) peptide adopts an a-helical conformation in solution. Moreover, NarJ recognizes and binds the helical NarG(1–15) peptide mostly via hydrophobic interactions as deduced from isothermal titration calorimetry analysis. NMR and differential scanning calorimetry analysis revealed a modification of NarJ conformation during complex formation with the NarG(1–15) peptide. Isothermal titration calo- rimetry and BIAcore experiments support a model whereby the protonated state of the chaperone controls the time dependence of peptide interaction. Structured digital abstract l MINT-7557484: NarJT (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) bind (MI:0407)by isothermal titration calorimetry ( MI:0065) l MINT-7557456: NarJT (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) bind (MI:0407)by nuclear magnetic resonance ( MI:0077) Abbreviations DSC, differential scanning calorimetry; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; k off, off rate constant; k on, on rate constant; TorA, trimethylamine N-oxide reductase. 1886 FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS with two distinct sites of the apoenzyme, one of them corresponding to the N-terminus of NarG, are respon- sible for the multifunctional character of NarJ [3,7]. NarJ binding on to this region represents part of a chaperone-mediated quality control process preventing membrane anchoring of the NarGH complex before all maturation events have been completed. This process strongly resembles the ‘Tat proofreading’ of periplasmic metalloproteins, of which the best-studied example relates to E. coli trimethylamine N-oxide reductase, TorA [8]. The targeting of this enzyme to the Tat translocase is prevented by the TorD chaper- one until the molybdenum cofactor has been inserted [8]. TorD binds the TorA signal peptide, thus shielding it from the Tat transporter [9,10]. Despite considerable research into chaperone function, only partial structural information has been gained on the nature and site of peptide interaction [9–12]. Biophysical studies have indicated that Tat signal peptides are unstructured in aqueous solution and acquire a high degree of secondary structure in hydrophobic environments, such as those that they may encounter upon interaction with their partners, either lipids from the cytoplasmic membrane or proteins such as chaperones or components of the Tat translocase [13,14]. Such a situation is encountered in the signal peptide of Sec substrates, which adopts an a-helical conformation in the SecA-bound state [15]. In the present study, the interaction between the NarJ chaperone and the N-terminus of NarG was studied using a series of biophysical approaches. In particular, NMR showed that the amphiphilic a-helix adopted by the N-terminus of NarG within the NarGHI complex [4] is conserved in NarG(1–15) and NarG(1–28) peptides. The docking calculation analysis revealed that NarG(1–15) interacts within a highly conserved elongated and hydrophobic groove of NarJ. Moreover, NMR and differential scanning calorimetry (DSC) revealed that upon peptide binding, NarJ undergoes a conformational change. Isothermal titra- tion calorimetry (ITC) and BIAcore analysis showed that protonation of the chaperone is responsible for a pH-dependent modulation of the peptide binding affinity. Results and Discussion The N-terminal part of the NarG subunit adopts a helical conformation in solution Our previous studies [3] revealed that the N-terminus of NarG is specifically targeted by NarJ during the matu- ration process. The X-ray structure of the NarGHI complex indicated that this region is made up of an amphiphilic helix (residues Ser2-Lys12) followed by an extended b-hairpin in close contact with both NarH and NarI subunits [4]. Here we addressed the question of the structure adopted by the N-terminus of NarG dur- ing the recognition process. At first, we synthesized two peptides [NarG(1–15) and NarG(1–28)] and solved their structures by NMR; NarG(1–15) corresponding to the predicted N-terminal helix and NarG(1–28), which included both the N-terminal helix and the b-sheet pres- ent in the mature NarGHI complex. The 1 H, 15 N-hetero- nuclear single quantum coherence (HSQC) spectra at pH 4.5 of both peptides and medium range NOEs were in agreement with the presence of an a-helix (residues Ser2-Phe11) in both peptides (Figs 1 and 2). At pH 7, the observed NH exchange was faster for NarG(1–15) than for NarG(1–28), indicating the presence of a less- structured N-terminal helix in the shorter peptide. These observations were confirmed by structure calculations of both peptides at pH 4.5 (Fig.3,Table 1). The struc- ture of NarG(1–28) consisted of an a-helix (residues 2–11) followed by an antiparallel pair of b-strands (residues 16–19 and 22–25). The N-terminal helix was similar to that observed in the NarG X-ray structure (rmsd = 2.84 A ˚ for the backbone) [4]. However, the orientation of secondary structure elements was rather different in the solution structure, probably due to the rearrangement of the N-terminal part of NarG inter- acting with both NarH and NarI subunits within the NarGHI complex. Second, 1 H, 15 N-HSQC of NarG(1–28) at natural abundance showed minor shifts upon NarJ binding (Fig. S1). These results suggest that the structural conformation adopted by the peptide in solution remains unchanged upon complex formation. Structural properties of the NarJ chaperone E. coli NarJ is a member of a large family of dedi- cated chaperones involved in the biogenesis of metalloproteins, including TorD, DmsD and YcdY [2]. Available 3D structures show a helical fold of all mem- bers of this large family [11,16,17]. The 1 H, 15 N-HSQC NMR spectra of NarJ were well resolved, indicating that the protein is mainly folded (Fig. S2). However, more than 60 of 271 expected peaks were missing in the NMR spectra. The unobserved residues are probably contained in one or several zones of the protein and their relative mobility is probably correlated to the unfructuous crystallization assays. In the absence of structural data for E. coli NarJ, a 3D model was built by homology modelling. Because of a lack of similarity, the 50 C-terminal amino acids were removed, resulting in a model of the truncated NarJ protein (NarJT; S. Zakian et al. Structural basis for peptide recognition by NarJ FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS 1887 Fig. S3). This structural model showed seven well- defined a-helices and confirms that NarJ belongs to the family of all a proteins. A similar truncated protein was constructed and we observed that the 60 previously missing signals remained absent in the 1 H, 15 N-HSQC spectrum of NarJT. These observations render it impossible to solve the structure of both NarJ and NarJT by NMR. 2D 1 H, 15 N-HSQC NMR spectra of both NarJ and NarJT were found to be very similar (Fig. S2). Moreover, thermal denaturation analysis of NarJ and NarJT carried out by DSC entailed a nontwo-state transition followed by irreversible processes. The temperature dependence of the partial molar heat capacity of both proteins was similar (Fig. 4A,B), indicating the exis- tence of only one structural domain on the protein. Upon peptide binding, NarJ undergoes a conformational change The temperature dependence of the partial molar heat capacity of free NarJ or NarJT differed considerably from that of their complexes with NarG(1–15) peptide. There was a marked increase in thermostability (10 °C) of both proteins due to peptide binding (Fig. 4A, B). Moreover, titration of the complex formation between the NarG(1–15) peptide and 15 N-labelled NarJT was monitored by 2D 1 H, 15 N-HSQC experiments. Spectrum analysis showed that most of the NarJ correlation peaks were affected upon peptide binding (Fig. 4C). The decrease in some of the free state resonances and the appearance of new resonances upon complex formation indicated a slow exchange on the NMR timescale between the free and the bound forms for NarJT. These results and the higher excess partial molar heat capacity of the complex observed by DSC are in agreement with a conformational change in NarJ upon interaction with both NarG peptides. NarJ ⁄ NarG complex formation is mostly entropy driven and undergoes pH-dependent modulation of the binding affinity To obtain more details about the interaction, ITC was used to monitor the binding of the NarG peptides to NarJ. Surprisingly, the binding isotherm was biphasic, with the best fit obtained with a two binding site model, comprising a first site with binding stoichiome- try (n) of 0.3 ± 0.2 and a binding constant (K d )of 3.4 ± 4 · 10 )9 m and a second with a stoichiometry of 0.7 ± 0.1 and a K d of 3.3 ± 3 · 10 )7 m (Fig. 5A). Identical results were obtained using NarJ or NarJT and both NarG peptides, allowing the delineation of a minimal complex formed between NarJT and the NarG(1–15) peptide (Table 2). Binding reactions are often coupled to the absorption or release of protons by the protein or the ligand. If this is the case, the bind- ing enthalpy is dependent on the ionization enthalpy of the buffer in which the reaction takes place. ITC exper- iments were therefore carried out in Hepes buffer having a different heat of ionization (20.5 kJÆmol )1 for Hepes and 47.4 kJÆmol )1 for the Tris ⁄ HCl used in the experiments reported in Table 2) and yielded an identi- cal biphasic isotherm with unmodified K d values. The enthalpy values obtained for the complex made between NarJ and any of the NarG peptides were lower than with Tris ⁄ HCl buffer ()38.8 ± 4 kJÆmol )1 in Hepes instead of )69.4 ± 3.8 kJÆmol )1 for Tris ⁄ HCl for the first site and )35 ± 3.6 kJÆmol )1 in Hepes instead of )62.1 ± 3.1 kJÆmol )1 for Tris ⁄ HCl for the A B Fig. 1. 1 H, 15 N-HSQC spectra of (A) NarG(1–15) and (B) NarG(1–28) peptides recorded at natural abundance on a 600 MHz NMR spec- trometer equipped with a cryoprobe. The experiments were recorded at 293 K using a 1 m M peptide sample concentration at pH 4.5. All residues are labelled according to the sequence. Structural basis for peptide recognition by NarJ S. Zakian et al. 1888 FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS second site). The measured enthalpy is the sum of two terms: the reaction enthalpy, independent of the buffer used in the experiment, and another term representing the contribution of the proton ionization of the buffer, which is multiplied by the number of protons that are absorbed (or released if negative) by the NarJ–peptide complex upon binding. On the basis of these experi- ments, we calculated a net release of approximately one proton during the binding process. Accounting for this, the results showed that the binding of NarG was mostly driven by positive entropy, although a negative enthalpy was also measured for both subpopulations (Table 2). Considering the increase in thermostability observed by DSC, the large and positive entropy was interpreted as the result of hydrophobic contacts or the loss of water-mediated hydrogen bonds. Interestingly, this biphasic behaviour disappeared by increasing the pH, suggesting a protonation event. At pH 8, the binding isotherm generated a sigmoidal binding curve that reached saturation with n =1.3±0.2andanapp- arent K d =1±1· 10 )7 m for NarJT ⁄ NarG(1–15) (Table 2, Fig. 5B). The pKa value of the protonable A B Fig. 3. Ensemble of the backbone traces of the 20 lowest energy conformers of the solution structure of (A) NarG(1–15) and (B) NarG(1–28). A B Fig. 2. (A) Sequences of NarG(1–15) (left) and NarG(1–28) (right) and sequential assignments. Collected sequential NOEs are classified into thick and thin bars according to their relative intensity. (B) NOE distribution versus sequence of NarG(1–15) (left) and NarG(1–28) (right). Intra- residual NOEs are in white, short NOEs are in light gray, medium-range NOEs are in dark gray, and long-range NOEs are in black. S. Zakian et al. Structural basis for peptide recognition by NarJ FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS 1889 residue that may be deduced from our data is lower than 7. Combining the DSC and ITC results, we con- clude that NarJ does not exhibit two binding sites, but rather exists as two distinct subpopulations, probably in rapid exchange in the free state. Each subpopulation binds the peptide with different affinities, but uses a similar overall mechanism. Protonation at or near the binding pocket may account for the existence of these two subpopulations. To assess the contribution of electrostatic interactions in NarJ peptide binding, we measured the energetics of complex formation in a buffer with a high salt concentration (500 mm NaCl). There was no effect on the binding constants (Table 2); however, the binding was purely entropy driven, indicating that hydrophobic interactions are responsible for the strong binding of NarG peptides to NarJ. To predict the interaction surface between NarJ and NarG, we performed a docking experiment in an ab initio mode using haddock software. Six of the 10 best clusters of docking solutions were located in a hydrophobic funnel-shaped cavity of the NarJT model (Fig. 6), confirming the hydrophobic character of the binding process predicted by ITC data. BIAcore surface plasmon resonance was used to investigate the kinetic parameters of the interaction (on rate constant k on and off rate constant k off ) between NarJ and the NarG(1–15) peptide. Taking into account the existence of two subpopulations of NarJ at pH 7, the BIAcore experimental data performed at the same pH were fitted with the heterogeneous ligand interaction Table 1. NMR and refinement statistics for NarG(1–15) and NarG(1–28) structures. NarG(1–15) NarG(1–28) NMR distance and dihedral constraints Total NOEs 243 536 Short range (|i)j| £ 1) 190 317 Medium range (1 < |i)j| < 5) 52 141 Long range (|i)j| ‡ 5) 1 78 Average pairwise rmsd a (A ˚ ) Heavy 2.24 ± 0.29 1.64 ± 0.19 Backbone 1.39 ± 0.32 0.94 ± 0.16 Ramachandran Most favoured and additional allowed (%) 100 96.2 Generously allowed (%) 0 3.8 Disallowed region (%) 0 0 a Calculated among 20 [NarG(1–15)] and 15 [NarG(1–28)] refined structures. AB C Fig. 4. Deconvolution of the transition excess heat capacity of (A) NarJ and (B) NarJT alone (black traces) or in complex with NarG(1–15) (red traces). Solid lines, experimental data; dotted lines, deconvolution peaks. NarJ 50.9 ± 1 °C; NarJ–NarG(1–15) 61.6 ± 1 °C; NarJT 53 ± 1 °C; NarJT–NarG(1–15) 63.2 ± 1 °C. (C) Overlay of 1 H, 15 N-HSQC spectra at 27 °C of NarJT in the absence (black trace) and in the presence (orange trace) of a 2 molar ratio of NarG(1–15). The experiments were recorded on a 500 MHz NMR spectrometer using a 0.1 m M sample concentration at pH 7. Structural basis for peptide recognition by NarJ S. Zakian et al. 1890 FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS model. The results indicated the existence of a minor population (27%) with a high affinity (K d = 4.4 ± 3 · 10 )9 m) and a major population (73%) with a lower affinity (K d =81±36· 10 )9 m). Analysis of the BIAcore experiments performed at pH 8 could only be fitted with the 1 : 1 Langmuir model of simple binding, confirming the existence of a single state of NarJ at this pH. These results are in full agreement with those obtained with ITC (Table 2). Interestingly, at pH 7, k off varied by nearly a factor of 10 between the two subpop- ulations, i.e. k off = 3.2 ± 1 s )2 for the minor species of high affinity and k off = 1.9 ± 1 s )1 for the major species of lower affinity. Overall, we concluded that protonation of a specific residue of NarJ modulates the peptide binding affinity, in particular via the lifespan of the protein–peptide complex. Conclusion One important finding is the structural flexibility of the NarJ chaperone and its conformational rearrangement upon NarG binding. Examination of the crystal structure of several members of this new family of chaperones [11,16,17] indicates the presence of several disordered regions. Moreover, ITC data obtained by others on E. coli TorD [9] and DmsD [12] have sys- tematically shown a strong decrease in entropy associ- ated with the complex formation. Overall, structural flexibility appears to be a common feature of this new family of chaperones. It is worth mentioning that the function of these proteins is not restricted to the recog- nition and binding of the N-terminus of the nascent metalloprotein, but includes their participation towards A B Fig. 5. Calorimetric titration of NarJ at (A) pH 7 or (B) pH 8 with NarG(1–15) in 50 m M Tris ⁄ HCl, 1 mM MgCl 2 , 100 mM NaCl. The upper panels show the raw data for the heat effect during the titrations; the lower panels are the binding isotherms. Table 2. Thermodynamic parameters of NarG(1–15) and NarG(1–28) peptides binding to NarJ and NarJT. The experiments were performed in 50 m M Tris ⁄ HCl pH 7, 1 mM MgCl 2 , 100 mM NaCl. The values presented are the average of at least three independent experiments. Complex nK d (M) DH (kJÆmol )1 ) DH corr a (kJÆmol )1 ) TDS (kJÆmol )1 ) TDS corr a (kJÆmol )1 ) NarJ–NarG(1–28) 0.2 ± 0.1 2.3 ± 4 · 10 )9 )69.4 ± 3.8 )22 )20.1 27.3 0.9 ± 0.1 1.7 ± 3.1 · 10 )7 )62.1 ± 3.1 )14.7 )23.4 24 NarJT–NarG(1–28) 0.3 ± 0.1 7.3 ± 3.8 · 10 )9 )57 ± 2.7 )9.6 )10.6 36.8 0.7 ± 0.2 1.9 ± 2.9 · 10 )7 )50.1 ± 3 )2.7 )11.8 35.6 NarJ–NarG(1–15) 0.3 ± 0.2 3.4 ± 4 · 10 )9 )56.4 ± 2.2 )9 )8.1 39.3 0.7 ± 0.1 3.3 ± 3 · 10 )7 )50.8 ± 2.6 )3.4 )13.8 33.6 NarJT–NarG(1–15) 0.3 ± 0.1 3.5 ± 2 · 10 )9 )43 ± 1.2 4.4 5.2 52.6 0.6 ± 0.2 2.5 ± 1.9 · 10 )7 )53.7 ± 1.1 )6.3 )16 31.4 NarJT–NarG(1–15) (At pH 8.0) 1.3 ± 0.2 1 ± 1 · 10 )7 )45.5 ± 0.4 1.9 )5.6 41.8 NarJ–NarG(1–15) (in 500 m M NaCl) 0.2 ± 0.1 10 ± 1 · 10 )9 )40.3 ± 2.3 7.1 5.3 52.7 1 ± 0.2 4.3 ± 2 · 10 )7 )29.2 ± 3.6 18.2 7.1 54.5 a Calculated after considering a net release of one proton according to the following equation: DH = DH corr +(nH + )DH ion , where DH corr is the true intrinsic heat of binding and nH + is the number of protons released or taken by the buffer upon binding (DH ion for Tris ⁄ HCl is 47.4 kJÆmol )1 ). S. Zakian et al. Structural basis for peptide recognition by NarJ FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS 1891 metal cofactor insertion processes through additional contacts with their specific partner [1]. Such structural flexibility may not only contribute to their high specific- ity during the binding process, but may also be of para- mount importance with regard to their multiple functions during the biogenesis of the partner. An exception would be the NapD chaperone having a fer- redoxin-type fold, which undergoes only minor confor- mational changes upon binding the twin-arginine signal peptide of NapA [18]. In this case, biogenesis of the NapA protein is assisted by NapF in charge of cofactor loading [19,20]. Overall, considering the global confor- mational change of the chaperone observed upon pep- tide binding, it is as essential to solve the structure of the chaperone–peptide complex as to evaluate quantita- tively the structural flexibility of the chaperone. An unexpected finding was the discovery of the pH-dependent modulation of the peptide binding affinity by changing the lifespan of the chaperone– peptide complex. Indeed, deprotonation of a yet unidentified residue of NarJ drastically reduces the pep- tide binding affinity by 100-fold and the lifespan of the complex by 10-fold, as judged by k off . The physiological chaperone cycle probably consists of the rapid binding of the N-terminus of the partner, regardless of whether it is a twin-arginine signal peptide or not, followed by its release once cofactor loading and protein folding are complete. Accordingly, we hypothesize that the proton- ated state of the chaperone initiates this cycle, whereas the deprotonated state occurs upon completion of the maturation process of the partner. The nature of the signal that may trigger dissociation of the complex remains unclear; however, we propose that a local perturbation of the hydrogen network surrounding the involved residue may alter its protonation state. Identification of the protonable residue clearly repre- sents a future challenge. Finally, we have demonstrated that the N-terminus of NarG, bearing some sequence similarity with twin- arginine peptides, adopts a helical conformation in solution, which remains largely unchanged upon NarJ binding. Overall, our studies should pave the way for future studies aiming to decipher the mechanism behind chaperone-mediated quality control. Experimental Procedures NarJ and NarJT production and purification Overexpression and purification of NarJ carrying a C-terminal hexahistidine tag were carried out as described previously using a pET22b derivative plasmid [21]. A new plasmidic construction where the coding region for the last 50 amino acids has been deleted from the abovementioned plasmid was made to allow overexpression of NarJT. Purifi- cation of NarJT was performed under the same conditions as NarJ. Isotopically labelled NarJ–His6 and NarJT–His6 proteins were produced using M9 minimum media and 15 N-labelled NH 4 Cl. N-terminal NarG peptides The NarG(1–15) MSKFLDRFRYFKQKG and NarG(1–28) MSKFLDRFRYFKQKGETFADGHGQLLNT peptides used in this study were chemically synthesized and purified by Synprosis (Marseilles, France). The molecular mass of each peptide was verified by mass spectrometry. NMR experiments for NarG peptide structure calculation NMR experiments were performed at 293 K, on a 1 mm peptide sample in 10 mm potassium phosphate buffer at pH 4.5. Homonuclear NOESY, TOCSY and COSY spectra and a 24 h 1 H, 15 N-HSQC spectrum at natural abundance were recorded for each peptide on a Bruker 600 MHz spec- trometer equipped with a TCN cryoprobe. Spectra were processed using the topspin 2.1 software (Bruker BioSpin S.A., Wissembourg Ce ´ dex, France). C-ter N-ter Fig. 6. Interaction surface between NarJT and the N-terminus of NarG predicted by ab initio docking experiments. The blue spheres represent the centre of geometry of the NarG(1–15) peptide. Only the best structures of each of the 10 best clusters are depicted ( HADDOCK score). Surface residues of NarJT in brown form the bottom of the funnel-shaped cavity, residues represented in light orange form the entry, whereas the rest are in orange. Structural basis for peptide recognition by NarJ S. Zakian et al. 1892 FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS Resonance assignment and NOE integration were obtained using cara software [22]. Peak volumes were automatically converted into upper-limit distances by the calibration routine of cyana 2.1 software [23]. In total, 100 structures were calculated per iteration and the 20 best structures of the last iteration were retained for water refinement using crystallography & NMR system [24]. Visual analysis of the final selected structures was carried out using pymol software [25] and the geometric quality of the resulting structures was assessed using procheck 3.4 and procheck-nmr [26]. ITC ITC was performed using an MCS ITC microcalorimeter (Microcal LLC, Northampton, MA, USA) at 298 K. The experimental data fitting was carried out using origin 7.0 (Origin Lab Corporation, Northampton, MA, USA). NarJ, NarJT and NarG peptides were dialysed in different buffers as indicated. The heat of dilution was measured by injecting the ligand into the protein-free buffer solution or by addi- tional injections of peptide after saturation. The obtained value was then subtracted from the heat of the reaction to obtain the effective heat of binding [27]. DSC Heat denaturation measurements were carried out on a MicroCal VP-DSC instrument (Microcal LLC) at a heating rate of 1 KÆmin )1 . The denaturation temperature was deter- mined as previously described [28]. Because of the irrevers- ibility of the denaturation process, the excess molar heat capacity of the protein could not be determined. BIAcore surface plasmon resonance analysis All experiments were carried out at 298 K on a BIAcore 3000 apparatus (BIAcore, GE Healthcare Europe GmbH, Orsay, France). NarJ–His6 was immobilized on a CM5 sensor chip using amine coupling [21]. NarG(1–15) peptide in 10 mm Tris ⁄ HCl, 150 mm NaCl, 3.4 mm EDTA, 0.005% surfactant P20 and pH 7 or 8 was then injected over the test and control (no protein immobilized) surfaces at a flow rate of 60 lLÆmin )1 . The sensor surface was regenerated with an injection of 1 mm NaOH final concentration. The resulting sensorgrams were evaluated using the biomolecu- lar interaction analysis evaluation software (BIAcore) to calculate the kinetic constants of the complex formation. Molecular docking A molecular model of NarJT was obtained using modeller software. Briefly, the NarJ sequence was first used to find related structures from the Protein Data Bank using the NCBI server Psi-Blast. To improve the overall quality of multiple alignments, 21 sequences related to NarJ from the NR databank were selected by a single Blast search from the NCBI server. These sequences were used to derive multiple structure–sequence alignments using the program t-coffee [29] (Fig. S4). These multiple structure–sequence alignments were used by the program modeller [30] to generate a set of 20 NarJT homology models with different spatial conformations. Docking experiments were carried out with haddock software [31] using the NarJT model and the NarG(1–15) structure. The dockings were run on the HADDOCK web server (http://haddock.chem.uu.nl/). Ab initio docking was performed using the solvated docking mode. The number of calculated structures in the rigid body step was set to 10 000; 200 structures were obtained after semiflexible and explicit solvent refinement steps. Acknowledgements We thank Drs G. Giordano and A. Walburger for criti- cal reading of the manuscript, A. Cornish-Bowden for stimulating discussions and revising the manuscript, O. Bornet for providing NMR experiments, G. Ferracci for BIAcore experiments and Angloscribe for revising. This work was supported by the CNRS, ANR (to AM, project BIODYNMET), IBiSA and Canceropole PACA. SZ was supported by a fellowship from the Conseil Re ´ gional PACA. AV was supported by a FRM fellowship. JBC was supported by a MESR fellowship. References 1 Magalon A & Mendel RR (2008) Biosynthesis and insertion of the molybdenum cofactor. In EcoSal—Escherichia coli and Salmonella: cellular and molecular biology (Bo ¨ ck A, Curtiss R III, Kaper JB, Karp PD, Neidhardt FC, Nystro ¨ m T, Slauch JM & Squires CL eds). ASM Press, Washington, DC. 2 Sargent F (2007) Constructing the wonders of the bacterial world: biosynthesis of complex enzymes. Microbiology, 153, 633–651. 3 Vergnes A, Pommier J, Toci R, Blasco F, Giordano G & Magalon A (2006) NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly. J Biol Chem, 281, 2170–2176. 4 Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH & Strynadka NC (2003) Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat Struct Biol, 10, 681–687. 5 Rothery RA, Blasco F, Magalon A & Weiner JH (2001) The diheme cytochrome b subunit (Narl) of S. Zakian et al. Structural basis for peptide recognition by NarJ FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS 1893 Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols. J Mol Microbiol Biotechnol 3, 273–283. 6 Blasco F, Guigliarelli B, Magalon A, Asso M, Giordano G & Rothery RA (2001) The coordination and function of the redox centres of the membrane-bound nitrate reductases. Cell Mol Life Sci 58, 179–193. 7 Lanciano P, Vergnes A, Grimaldi S, Guigliarelli B & Magalon A (2007) Biogenesis of a respiratory complex is orchestrated by a single accessory protein. J Biol Chem, 282, 17468–17474. 8 Jack RL, Buchanan G, Dubini A, Hatzixanthis K, Palmer T & Sargent F (2004) Coordinating assembly and export of complex bacterial proteins. EMBO J, 23, 3962–3972. 9 Hatzixanthis K, Clarke TA, Oubrie A, Richardson DJ, Turner RJ & Sargent F (2005) Signal peptide-chaperone interactions on the twin-arginine protein transport pathway. Proc Natl Acad Sci USA, 102, 8460–8465. 10 Buchanan G, Maillard J, Nabuurs SB, Richardson DJ, Palmer T & Sargent F (2008) Features of a twin- arginine signal peptide required for recognition by a Tat proofreading chaperone. FEBS Lett, 582, 3979– 3984. 11 Kirillova O, Chruszcz M, Shumilin IA, Skarina T, Gorodichtchenskaia E, Cymborowski M, Savchenko A, Edwards A & Minor W (2007) An extremely SAD case: structure of a putative redox-enzyme maturation protein from Archaeoglobus fulgidus at 3.4 A resolution. Acta Crystallogr D Biol Crystallogr, 63, 348–354. 12 Winstone TL, Workentine ML, Sarfo KJ, Binding AJ, Haslam BD & Turner RJ (2006) Physical nature of signal peptide binding to DmsD. Arch Biochem Biophys, 455, 89–97. 13 Kipping M, Lilie H, Lindenstrauss U, Andreesen JR, Griesinger C, Carlomagno T & Bruser T (2003) Struc- tural studies on a twin-arginine signal sequence. FEBS Lett, 550, 18–22. 14 San Miguel M, Marrington R, Rodger PM, Rodger A & Robinson C (2003) An Escherichia coli twin-arginine signal peptide switches between helical and unstructured conformations depending on the hydrophobicity of the environment. Eur J Biochem , 270, 3345–3352. 15 Gelis I, Bonvin AM, Keramisanou D, Koukaki M, Gouridis G, Karamanou S, Economou A & Kalodimos CG (2007) Structural basis for signal-sequence recogni- tion by the translocase motor SecA as determined by NMR. Cell, 131, 756–769. 16 Tranier S, Iobbi-Nivol C, Birck C, Ilbert M, Mortier-Barriere I, Mejean V & Samama JP (2003) A novel protein fold and extreme domain swapping in the dimeric TorD chaperone from Shewanella massilia. Structure, 11, 165–174. 17 Qiu Y, Zhang R, Binkowski TA, Tereshko V, Joachimiak A & Kossiakoff A (2008) The 1.38 A crystal structure of DmsD protein from Salmonella typhimurium, a proofreading chaperone on the Tat pathway. Proteins, 71, 525–533. 18 Maillard J, Spronk CA, Buchanan G, Lyall V, Richardson DJ, Palmer T, Vuister GW & Sargent F (2007) Structural diversity in twin-arginine signal peptide-binding proteins. Proc Natl Acad Sci USA, 104, 15641–15646. 19 Olmo-Mira MF, Gavira M, Richardson DJ, Castillo F, Moreno-Vivian C & Roldan MD (2004) NapF is a cytoplasmic iron-sulfur protein required for Fe-S cluster assembly in the periplasmic nitrate reductase. J Biol Chem, 279, 49727–49735. 20 Nilavongse A, Brondijk TH, Overton TW, Richardson DJ, Leach ER & Cole JA (2006) The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA. Microbiology, 152, 3227–3237. 21 Blasco F, Dos Santos JP, Magalon A, Frixon C, Guigliarelli B, Santini CL & Giordano G (1998) NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. Mol Microbiol, 28, 435–447. 22 Keller R (2004) The computer aided resonance assignment tutorial. 112-113. Cantina Verlag, Goldau. 23 Guntert P (2004) Automated NMR structure calcula- tion with CYANA. Methods Mol Biol, 278, 353–378. 24 Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al. (1998) Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr, 54, 905–921. 25 DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA. 26 Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R & Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR, 8, 477–486. 27 Makarov AA, Tsvetkov PO, Villard C, Esquieu D, Pourroy B, Fahy J, Braguer D, Peyrot V & Lafitte D (2007) Vinflunine, a novel microtubule inhibitor, suppresses calmodulin interaction with the microtubule- associated protein STOP. Biochemistry 46, 14899–14906. 28 Privalov PL & Potekhin SA (1986) Scanning microcal- orimetry in studying temperature-induced changes in proteins. Methods Enzymol, 131, 4–51. 29 Notredame C, Higgins DG & Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol, 302, 205–217. Structural basis for peptide recognition by NarJ S. Zakian et al. 1894 FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS 30 Sali A & Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol, 234, 779–815. 31 Dominguez C, Boelens R & Bonvin AM (2003) HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc, 125, 1731–1737. Supporting information The following supplementary material is available: Fig. S1. Overlay of 1 H, 15 N-HSQC spectra at 300 K of NarG(1–28) peptide in the absence (black trace) and in the presence (orange trace) of 2 molar ratio of NarJ. Fig. S2. Overlay of 1 H, 15 N-HSQC spectra at 300 K of NarJ (black) and NarJT (orange). Fig. S3. Ribbon diagram of the NarJT model. The structures are displayed using pymol. Fig. S4. Multiple structure–sequence alignment of the 25 sequences used for the homology modelling of NarJT protein produced with t-coffee. 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. S. Zakian et al. Structural basis for peptide recognition by NarJ FEBS Journal 277 (2010) 1886–1895 ª 2010 The Authors Journal compilation ª 2010 FEBS 1895 . Basis of recognition between the NarJ chaperone and the N-terminus of the NarG subunit from Escherichia coli nitrate reductase Silva Zakian 1 ,. previously shown that the NarJ chaperone interacts with the N-terminus of the NarG subunit coming from the nitrate reductase complex, NarGHI. In the present study,