Tài liệu Báo cáo khoa học: Basis of recognition between the NarJ chaperone and the N-terminus of the NarG subunit from Escherichia coli nitrate reductase pdf

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:
(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 ( />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.
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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,
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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