Basis of recognition between the NarJ chaperone and the N-terminus of the NarG subunit from Escherichia coli nitrate reductase


  • Silva Zakian,

    1.  Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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  • Daniel Lafitte,

    1.  MaP site Timone, UMR INSERM 911, Université d’Aix-Marseille II, France
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  • Alexandra Vergnes,

    1.  Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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  • Cyril Pimentel,

    1.  Interactions et Modulateurs de Réponses, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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  • Corinne Sebban-Kreuzer,

    1.  Interactions et Modulateurs de Réponses, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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  • René Toci,

    1.  Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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  • Jean-Baptiste Claude,

    1.  Information Génomique et Structurale, Marseille, France
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  • Françoise Guerlesquin,

    1.  Interactions et Modulateurs de Réponses, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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  • Axel Magalon

    1.  Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique, Marseille, France
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A. Magalon, Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, 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


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 α-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 calorimetry and BIAcore experiments support a model whereby the protonated state of the chaperone controls the time dependence of peptide interaction.

Structured digital abstract


differential scanning calorimetry


heteronuclear single quantum coherence


isothermal titration calorimetry


off rate constant


on rate constant


trimethylamine N-oxide reductase


A new family of molecular chaperones, conserved in most prokaryotes, performs essential roles in the biogenesis of both exported and nonexported metalloproteins [1,2]. They share a common fold composed entirely of α-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 nonexported 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 with two distinct sites of the apoenzyme, one of them corresponding to the N-terminus of NarG, are responsible 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 chaperone 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 α-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 α-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 titration 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 maturation 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 β-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 during 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 β-sheet present in the mature NarGHI complex. The 1H,15N-heteronuclear single quantum coherence (HSQC) spectra at pH 4.5 of both peptides and medium range NOEs were in agreement with the presence of an α-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 structure of NarG(1–28) consisted of an α-helix (residues 2–11) followed by an antiparallel pair of β-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 Å 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 interacting with both NarH and NarI subunits within the NarGHI complex. Second, 1H,15N-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.

Figure 1.

1H,15N-HSQC spectra of (A) NarG(1–15) and (B) NarG(1–28) peptides recorded at natural abundance on a 600 MHz NMR spectrometer equipped with a cryoprobe. The experiments were recorded at 293 K using a 1 mm peptide sample concentration at pH 4.5. All residues are labelled according to the sequence.

Figure 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). Intraresidual NOEs are in white, short NOEs are in light gray, medium-range NOEs are in dark gray, and long-range NOEs are in black.

Figure 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).

Table 1.   NMR and refinement statistics for NarG(1–15) and NarG(1–28) structures.
  1. aCalculated among 20 [NarG(1–15)] and 15 [NarG(1–28)] refined structures.

NMR distance and dihedral constraints
 Total NOEs243536
 Short range (|ij| ≤ 1)190317
 Medium range (1 < |ij| < 5)52141
 Long range (|ij| ≥ 5)178
Average pairwise rmsda (Å)
 Heavy2.24 ± 0.291.64 ± 0.19
 Backbone1.39 ± 0.320.94 ± 0.16
 Most favoured and additional allowed (%)10096.2
 Generously allowed (%)03.8
 Disallowed region (%)00

Structural properties of the NarJ chaperone

E. coli NarJ is a member of a large family of dedicated chaperones involved in the biogenesis of metalloproteins, including TorD, DmsD and YcdY [2]. Available 3D structures show a helical fold of all members of this large family [11,16,17]. The 1H,15N-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; Fig. S3). This structural model showed seven well-defined α-helices and confirms that NarJ belongs to the family of all α proteins.

A similar truncated protein was constructed and we observed that the 60 previously missing signals remained absent in the 1H,15N-HSQC spectrum of NarJT. These observations render it impossible to solve the structure of both NarJ and NarJT by NMR. 2D 1H,15N-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 existence of only one structural domain on the protein.

Figure 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 1H,15N-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 mm sample concentration at pH 7.

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 15N-labelled NarJT was monitored by 2D 1H,15N-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 stoichiometry (n) of 0.3 ± 0.2 and a binding constant (Kd) of 3.4 ± 4 × 10−9 m and a second with a stoichiometry of 0.7 ± 0.1 and a Kd 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 binding enthalpy is dependent on the ionization enthalpy of the buffer in which the reaction takes place. ITC experiments were therefore carried out in Hepes buffer having a different heat of ionization (20.5 kJ·mol−1 for Hepes and 47.4 kJ·mol−1 for the Tris/HCl used in the experiments reported in Table 2) and yielded an identical biphasic isotherm with unmodified Kd 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 kJ·mol−1 in Hepes instead of −69.4 ± 3.8 kJ·mol−1 for Tris/HCl for the first site and −35 ± 3.6 kJ·mol−1 in Hepes instead of −62.1 ± 3.1 kJ·mol−1 for Tris/HCl for the 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 experiments, 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 = 1.3 ± 0.2 and an apparent Kd = 1 ± 1 × 10−7 m for NarJT/NarG(1–15) (Table 2, Fig. 5B). The pKa value of the protonable residue that may be deduced from our data is lower than 7. Combining the DSC and ITC results, we conclude 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.

Figure 5.

 Calorimetric titration of NarJ at (A) pH 7 or (B) pH 8 with NarG(1–15) in 50 mm Tris/HCl, 1 mm MgCl2, 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 mm Tris/HCl pH 7, 1 mm MgCl2, 100 mm NaCl. The values presented are the average of at least three independent experiments.
ComplexnKd (m)ΔH (kJ·mol−1)ΔHcorra (kJ·mol−1)TΔS (kJ·mol−1)TΔScorra (kJ·mol−1)
  1. a Calculated after considering a net release of one proton according to the following equation: ΔH = ΔHcorr + (nH+Hion, where ΔHcorr is the true intrinsic heat of binding and nH+ is the number of protons released or taken by the buffer upon binding (ΔHion for Tris/HCl is 47.4 kJ·mol−1).

NarJ–NarG(1–28)0.2 ± 0.12.3 ± 4 × 10−9−69.4 ± 3.8−22−20.127.3
0.9 ± 0.11.7 ± 3.1 × 10−7−62.1 ± 3.1−14.7−23.424
NarJT–NarG(1–28)0.3 ± 0.17.3 ± 3.8 × 10−9−57 ± 2.7−9.6−10.636.8
0.7 ± 0.21.9 ± 2.9 × 10−7−50.1 ± 3−2.7−11.835.6
NarJ–NarG(1–15)0.3 ± 0.23.4 ± 4 × 10−9−56.4 ± 2.2−9−8.139.3
0.7 ± 0.13.3 ± 3 × 10−7−50.8 ± 2.6−3.4−13.833.6
NarJT–NarG(1–15)0.3 ± 0.13.5 ± 2 × 10−9−43 ±
0.6 ± 0.22.5 ± 1.9 × 10−7−53.7 ± 1.1−6.3−1631.4
NarJT–NarG(1–15) (At pH 8.0)1.3 ± 0.21 ± 1 × 10−7−45.5 ± 0.41.9−5.641.8
NarJ–NarG(1–15) (in 500 mm NaCl)0.2 ± 0.110 ± 1 × 10−9−40.3 ±
1 ± 0.24.3 ± 2 × 10−7−29.2 ± 3.618.27.154.5

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.

Figure 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.

BIAcore surface plasmon resonance was used to investigate the kinetic parameters of the interaction (on rate constant kon and off rate constant koff) 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 model. The results indicated the existence of a minor population (27%) with a high affinity (Kd= 4.4 ± 3 × 10−9 m) and a major population (73%) with a lower affinity (Kd = 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, koff varied by nearly a factor of 10 between the two subpopulations, i.e. koff = 3.2 ± 1 s−2 for the minor species of high affinity and koff = 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.


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 systematically shown a strong decrease in entropy associated 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 recognition and binding of the N-terminus of the nascent metalloprotein, but includes their participation towards metal cofactor insertion processes through additional contacts with their specific partner [1]. Such structural flexibility may not only contribute to their high specificity during the binding process, but may also be of paramount importance with regard to their multiple functions during the biogenesis of the partner. An exception would be the NapD chaperone having a ferredoxin-type fold, which undergoes only minor conformational 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 conformational change of the chaperone observed upon peptide binding, it is as essential to solve the structure of the chaperone–peptide complex as to evaluate quantitatively 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 peptide binding affinity by 100-fold and the lifespan of the complex by 10-fold, as judged by koff. 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 protonated 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 represents 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. Purification 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 15N-labelled NH4Cl.

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 1H,15N-HSQC spectrum at natural abundance were recorded for each peptide on a Bruker 600 MHz spectrometer equipped with a TCN cryoprobe. Spectra were processed using the topspin 2.1 software (Bruker BioSpin S.A., Wissembourg Cédex, France).

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 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 additional 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].


Heat denaturation measurements were carried out on a MicroCal VP-DSC instrument (Microcal LLC) at a heating rate of 1 K·min−1. The denaturation temperature was determined as previously described [28]. Because of the irreversibility 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 μL·min−1. The sensor surface was regenerated with an injection of 1 mm NaOH final concentration. The resulting sensorgrams were evaluated using the biomolecular 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 ( 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.


We thank Drs G. Giordano and A. Walburger for critical 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 Régional PACA. AV was supported by a FRM fellowship. JBC was supported by a MESR fellowship.