Tải bản đầy đủ

Tài liệu Báo cáo khóa học: The C-terminal domain of Escherichia coli Hfq increases the stability of the hexamer ppt

The C-terminal domain of
Escherichia coli
Hfq increases the stability
of the hexamer
ronique Arluison
, Marc Folichon
, Sergio Marco
, Philippe Derreumaux
, Olivier Pellegrini

me Seguin
, Eliane Hajnsdorf
and Philippe Regnier
Institut de Biologie Physico-Chimique CNRS UPR 9073 conventionne
e avec l’universite
Paris 7, Paris, France;
Institut Curie CNRS
UMR 168, Paris, France;
Institut de Biologie Physico-Chimique CNRS UPR 9080, Paris, France;
Service de Biophysique des
Fonctions Membranaires, DBJC/CEA & URA 2096 CNRS, Gif/Yvette, France
The Hfq (Host factor 1) polypeptide is a nucleic acid binding
protein involved in the synthesis of many polypeptides.
Hfq particularly affects the translation and the stability of
several RNAs. In an earlier study, the use of fold recog-
nition methods allowed us to detect a relationship between
Escherichia coli Hfq and the Sm topology. This topology
was further validated by a series of biophysical studies and
the Hfq structure was modelled on an Sm protein. Hfq forms
a b-sheet ring-shaped hexamer. As our previous study pre-
dicted a large number of alternative conformations for the
C-terminal region, we have determined whether the last 19
C-terminal residues are necessary for protein function. We
find that the C-terminal truncated protein is fully capable of
binding a polyadenylated RNA (K
of 120 p
vs. 50 p
full-length Hfq). This result shows that the functional core of
E. coli Hfq resides in residues 1–70 and confirms previous
genetic studies. Using equilibrium unfolding studies, how-
ever, we find that full-length Hfq is 1.8 kcalÆmol
stable than its truncated variant. Electron microscopy ana-
lysis of both truncated and full-length proteins indicates a
structural rearrangement between the subunits upon trun-
cation. This conformational change is coupled to a reduction
in b-strand content, as determined by Fourier transform
infra-red. On the basis of these results, we propose that
the C-terminal domain could protect the interface between
the subunits and stabilize the hexameric Hfq structure. The
origin of this C-terminal domain is also discussed.
Keywords: RNA binding protein; Sm-like (L-Sm); b-topol-
ogy; urea equilibrium unfolding; electron microscopy.
Hfq (Host factor 1) of Escherichia coli is an 11 kDa
polypeptide which was originally discovered as a host factor
required for the replication of bacteriophage Qb RNA [1].
However, by inactivating of the Hfq gene, it was later
demonstrated that it is involved in a variety of other
metabolic pathways [2–4]. In particular, Hfq has been
implicated in the translation and the control of the stability
of certain mRNAs. For example, Hfq has been shown
to interfere directly with ribosome binding of the ompA
transcript, exposing the transcript to ribonucleases [5,6]. It
has also been implicated in the stimulation of the elongation
of poly(A) tails by poly(A) polymerase, leading to poly(A)-
dependent mRNA degradation [7]. Finally, it has been
shown to be involved in the translation regulation of the
rpoS transcript, encoding the r
subunit of RNA poly-
merase and, as a consequence, influences the expression of
many stationary phase genes whose transcription depends
on r
[3]. This last effect was the first cellular role observed
for Hfq and has since been the subject of much attention
because Hfq influences rpoS translation by altering the
binding of small RNAs (sRNAs) to their complementary
target sequence [8–11]. The sRNAs involved in rpoS
translation control are OxyS, DsrA, RprA. More recently,
it has been also shown that many other sRNA can interact
with Hfq, pointing to a global role of the protein in
facilitating sRNA function [12,13].
Little is known about the mechanism of Hfq action. It has
been shown to bind strongly to single-stranded RNAs that
are A and U rich. Taking into account its ability to rescue
a folding trap of a splicing defective intron [14] and its
requirement for the activity of many sRNAs [11,15], it has
been proposed to be an RNA chaperone. The interaction
between Hfq and RNA may increase the propensity of
RNA to interact with itself or other RNAs, but also its
susceptibility to nucleases or poly(A) polymerase.
Recently, the Sm-like nature of Hfq was proposed on the
basis of weak sequence similarities between the N-terminal
domain of Hfq and the Sm and Sm-like (L-Sm) proteins
of eukaryotes and archaea [11,15,16]. These proteins are
components of the spliceosome complex and are also
involved in other RNA metabolism steps [17,18]. The
relationship between Hfq and the Sm topology was further
confirmed by using fold recognition methodology and by a
series of biophysical and biochemical studies. The structure
of Hfq from E. coli was modelled on an Sm protein [16] and
Correspondence to Philippe Regnier, Institut de Biologie Physico-
Chimique CNRS UPR 9073 conventionne
e avec l’universite
Paris 7,
13 rue P. et M. Curie, 75005 Paris, France.
Fax: + 33 1 58 41 50 20, Tel.: + 33 1 58 41 51 32,
E-mail: regnier@ibpc.fr
Abbreviations: ATR-FTIR, attenuated total reflectance fourier
transform infra-red; EM, electron microscopy; Hfq
, E. coli Hfq;
, Hfq full-length; Hfq
, Hfq lacking the 19 last amino acid;
sRNAs, small RNAs.
(Received 19 December 2003, revised 2 February 2004,
accepted 6 February 2004)
Eur. J. Biochem. 271, 1258–1265 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04026.x
the model further confirmed by determination of the X-ray
structure of Staphylococcus aureus and E. coli Hfq proteins
[19,20]. Hfq forms a hexameric ring shaped structure
[11,15,16,19], essentially b-sheet in character, with a cationic
inner hole implicated in RNA binding [19]. However, the
crystal structures available for two Hfq proteins are
restricted to the N-terminal part of the protein (% 60 amino
acids): the short C-terminal region of S. aureus Hfq was
disordered and not resolved in its crystal structure and the
C-terminal region of E. coli was genetically removed for its
crystallization [19,20]. In addition, the last 26 amino acids
were not present in the modelled E. coli Hfq structure,
because the secondary structure prediction and fold-recog-
nition methods proposed a large variety of conformations
for this region. To investigate the impact of the C-terminal
part on Hfq structure and function, we removed the 19 C-
terminal residues proteolytically. RNA binding properties,
electron microscopy and urea unfolding analysis of the
truncated protein are presented in this paper. They indicate
that the C-terminal region has a structural and thermo-
dynamic role in stabilizing the hexameric form, but does
not affect the binding of a polyadenylated RNA.
Materials and methods
Unless otherwise specified, all enzymes and chemicals were
either from Sigma or Merck-Biochemicals.
Purification of Hfq
C-terminal His-tagged Hfq was purified from the
BL21(DE3) strain transformed with plasmid pTE607 as
follows: cells from the induced culture were resuspended in
20 mL of buffer containing 20 m
Tris/HCl, pH 7.8, 0.5
NaCl, 10% (v/v) glycerol and 0.1% (v/v) Triton X-100
at 4 °C. The suspension was passed through a French
press (1200 bar) and centrifuged for 30 min at 15 000 g.
Imidazole-HCl (pH 7.8) was added to the supernatant to
reach a final concentration of 1 m
. The resulting
suspension was applied to a 1 mL Ni
acid column (Qiagen). The resin was then washed
sequentially with % 15 column volumes of: (a) 20 m
Tris/HCl, pH 7.8 buffer containing 0.3
NaCl and
20 m
imidazole and (b) 50 m
sodium phosphate,
pH 6.0 buffer containing 0.3
NaCl, and Hfq was finally
eluted with a buffer containing 50 m
sodium phosphate,
pH 6.0, 0.3
NaCl and 250 m
imidazole. The fractions
containing Hfq were analysed by SDS/PAGE, pooled
andheatedto80°C for 15 min. Insoluble material was
removed by centrifugation and the supernatant was
dialysed in buffer containing 50 m
Tris/HCl, pH 7.5,
EDTA, 50 m
Cl, 5% (v/v) glycerol and 0.1%
(v/v) Triton X-100. The protein was stored at 4 °C.
Protein concentrations were determined by measuring the
absorption at 280 nm (e
at 1 mgÆmL
¼ 0.34) or by
using the Bradford assay (Bio-Rad) [21] with bovine
serum albumin as a standard.
Limited proteolysis and mass spectra analysis
Digestion of Hfq by chymotrypsin (ROCHE biochemi-
cals, Switzerland) was performed at 20 °Cin50m
Tris/HCl, pH 8 containing 100 m
NaCl. Chymotrypsin
(1 ng) was used for 1 lg of Hfq (concentration
, 200 lL). Aliquots of 20 lL were withdrawn
at different times and the reaction was stopped by adding
the universal protease inhibitor a
-macroglobulin (Roche
Biochemicals) at the same final concentration as that
of the enzyme. Digestion was monitored on 16.5%
Tris/tricine SDS/PAGE [22] without a ÔspacerÕ gel. The
N-terminal fragment (Hfq
) generated by chymotrypsin
(% 9 kDa) was purified by extensive dialysis (molecular
mass cut-off 3500 Da) against 10 m
Tris/HCl, pH 8,
buffer containing 80 m
NaCl, 1% (v/v) glycerol, 0.01%
(w/v) dodecyl-b-
-maltoside. The separation of the
C-terminal fragment (% 2 kDa) from the the N-terminal
fragment was verified by SDS/PAGE.
For mass spectrometry analyses, samples were desalted
using Zip Tips C4 (Millipore), as described in the technical
manual. MALDI-TOF mass spectra were recorded with a
Voyager STR-DE (Perspective Biosystems Inc., Framing-
ham, MA, USA) mass spectrometer equipped with a delayed
extraction device. Samples were recorded either in positive
reflectron or linear mode. Calibration was performed using
standards. For electrospray mass spectra, the sample was
dissolved in a H
O/acetonitrile/acetic acid mix (49 : 50 : 1;
v/v/v) and the spectrum was acquired in positive mode on
a QTOF II mass spectrometer (Micromass). A reconstructed
spectrum from multiply charged ions was obtained using the
MaxEnt algorithm (Max Ent I, Micromass).
Electron microscopy and image analysis
Aliquots of full-length (Hfq
) and proteolysed Hfq (Hfq
were adsorbed to 400 mesh carbon-coated grids and stained
with 1% (w/v) uranyl acetate. Samples were observed with
a Philips CM120 electron microscope at an accelerating
voltage of 120 kV and nominal magnification of 75 000·.
Images with a final pixel size of 0.26 nm were recorded using
a GATAN ssCCD camera. A total number of 3057 single
particles from Hfq
, and 4833 from Hfq
, were windowed
software [23] before classifica-
tion by a self-organizing Kohonen neural network [24].
Average and standard deviation images of the major
groups, from each protein sample, were computed and,
after performing a rotational power spectra analysis, a
sixfold symmetry was imposed. Resolution was estimated to
18 A
using the SSNR method [25]. Once the image was
filtered at the computed resolution and sixfold symmetry
imposed, a differential map between the proteolysed and
nonproteolysed Hfq average images was calculated by using
the student-t algorithm as implemented in the
software with a significance level a ¼ 0.05.
Gel-shift assay
Labelled RNA fragments (2.5 p
) were incubated with Hfq
for 20 min at 37 °Cin50lLof10m
Tris/HCl, pH 8
buffer containing 80 m
NaCl, 1% (v/v) glycerol, 0.01%
(w/v) dodecyl-b-
-maltoside. The RNA used consisted of
rpsO mRNA with a 18 nucleotide poly(A) tail [7]. The RNA
was titrated with an excess of Hfq, its concentration ranging
from 15 p
to 10 n
[26]. Complexes were separated on
native PAGE as described in Zhang et al. [27].
Ó FEBS 2004 The role of the C-terminal domain on Hfq (Eur. J. Biochem. 271) 1259
Infrared spectra
Attenuated total reflectance (ATR)-FTIR spectra of Hfq
[200 l
, previously dialysed overnight against 10 m
HCl, pH 8 containing 80 m
NaCl and 0.01% (w/v)
-maltoside] were measured with a Bruker vector
22 spectrophotometer equipped with a 45

attachment at 20 °C. The spectra are the average of 125
scans. Spectra were corrected for the linear dependence of
the absorption measured by ATR on the wavelength.
The water signal was removed by subtraction of a buffer
spectrum. Analysis of the protein secondary structure
composition within each Hfq was performed by deconvo-
lution of the absorption spectrum as a sum of Gaussian
components [28].
Equilibrium unfolding study
Unfolding of the hexameric Hfq protein in urea was
monitored with a PTI A1010 fluorimeter using the intrinsic
tyrosine fluorescence of Hfq. Protein concentration was
0.1 mgÆmL
) 1
. Tyrosine residues were excited at 275 nm
and emission monitored at 303 nm using a cell path-length
of 1 cm. A 5 min incubation in urea was required to reach
equilibrium. Analysis of denaturation curves was performed
as described in [29]. The free energy of unfolding, DG
to equation:
¼ DG
À m½urea;
where, DG
is the free energy of unfolding in water and
m represents the effectiveness of the denaturant in destabi-
lizing the protein.
The effect of urea on the quaternary structure of Hfq was
determined by rate zonal centrifugation. The centrifugation
was performed at 20 °C, overnight, at 40 000 r.p.m., in the
70 Ti rotor of a Beckman LE-80 ultracentrifuge. Hfq was
denatured in a 50 m
Tris/HCl pH 8 buffer containing
100 m
NaCl and 6
urea prior to centrifugation.
Samples (100 lL) of 10 l
of 25 mL 1–5% (w/v) saccharose gradients prepared in the
buffer with or without urea. Fractions (1 ml) were collected
and Hfq polypeptides precipitated with 1 vol. of cold 10%
trichloroacetic acid were detected by dot-blot using anti-Hfq
Ig. Gradients were calibrated using mass standards indica-
ted (Fig. 4 legend).
Two peptides were generated upon limited proteolysis
of Hfq by chymotrypsin, whose masses were determined
by ESI- and MALDI-TOF spectrometry. These masses,
together with the location of aromatic amino acids in the
sequence, allowed us to deduce that chymotrypsin cleaves
after Tyr83, while all other aromatic residues (Phe11, 39,
42 and Tyr25, 55) of the N-terminal domain are not
accessible to the protease. This cleavage generates an 83
amino acid fragment containing the N-terminal domain
conserved in bacterial Hfqs (% 65 amino acids in Hfq
and a short part of the C-terminal domain which is only
found in a limited number of Hfqs (Fig. 1). We used
the truncated polypeptide generated by chymotrypsin to
investigate whether Hfq lacking the C-terminal domain
exhibits identical structural and RNA binding properties
than the full-length protein.
Electron microscopy analysis of Hfq
and Hfq
performed in order to determine the effect of the C-terminal
domain truncation on Hfq structure. Rotational power
spectra analysis performed on average images demonstrated
a sixfold symmetry for each Hfq multimer (data not shown).
This analysis indicates that the C-terminal domain is not
necessary for hexamerization of the protein. Once the
symmetry was imposed, the average of the images present a
different shape, with a stain-excluding region with a % 70 A
maximum diameter (Fig. 2A) and a central stained region
having % 20 A
diameter. Indeed, the shape of full-length
Hfq appeared sharper than the proteolysed one (Fig. 2B).
The absolute value of the difference calculated from the two
average images (a ¼ 0.05) showed that Hfq
and Hfq
differ in 12 regions arranged in the form of two crowns
(Fig. 2C, top). The difference corresponding to the external
crown appeared positive when the full-length minus that
proteolysed is calculated (Fig. 2C, center-left), indicating
the presence of a stain-excluding region, which can be
assigned to the C-terminal domain not present in the
proteolysed protein. This domain is at the origin of the
sharpest shape of full-length Hfq (Fig. 2C, bottom-left) and
extend beyond the canonical Sm fold. It should however
be noted that these represents almost 20% of the subunit
mass and the amount of density in this feature is small. It is
thus probable that most of the peptide is not seen, because
it is not contrasted by the negative stain.
Interestingly, another difference is located on the internal
circumference that appears positive when the images of
proteolysed minus full-length were calculated (Fig. 2C,
center-right). This suggests a conformational change at the
interface between the subunits (Fig. 2C, bottom-right), that
have been described as the strand b4ofchainBandthe
strand b5 of chain A in the crystal structure [19,20]. As the
electron microscopy data pointed to a conformational
change at the subunits’ interface upon truncation of Hfq,
we measured the secondary structure composition of full-
length and truncated Hfq using infrared spectroscopy.
FTIR spectra showed that full-length Hfq has 34 ± 2
residues in b-strands and 10 ± 1 residues in a-helices, while
truncated Hfq has 28 ± 2 residues in b-strands and 9 ± 1
residues in helical conformation. Thus, protease cleavage
causes a loss of six residues in b-sheet residues in the
truncated protein. The margin of error in determining
secondary structures by FTIR being of the order of 2% in
comparing two similars (the error comes only from the
deconvolution method), the 6% overall difference in signal
is thus significant. We expect that this reduction in b-sheet
character affects six amino acids located in the N-terminal
region (i.e. the Sm-like domain) for three reasons: (a) the
C-terminal region of E. coli Hfq is flexible in the molecular
model [16], (b) that of S. aureus Hfq is disordered in the
crystal structure [19] and (c) the FTIR spectrum of the
C-terminal peptide is strongly dominated by a broad band
at 1641 cm
, indicating an unordered structure.
The ability of the conserved N-terminal fragment to bind
polyadenylated rpsO mRNA alone was tested using gel-
shift assays. Figure 3 shows the binding curves of Hfq
1260 V. Arluison et al. (Eur. J. Biochem. 271) Ó FEBS 2004
. The corresponding equilibrium dissociation con-
stants (K
) were found to be 50 ± 10 p
and 120 ± 15 p
for Hfq and Hfq
, respectively. The minor increase in
upon truncation suggests that the C-terminal domain
of Hfq does not contribute significantly, if at all, to poly-
adenylated RNA binding.
As no major effect on RNA binding could be attributed
to the C-terminal region, we determined the contribution of
this region to the thermodynamic stability of Hfq. Equilib-
rium unfolding studies of Hfq were performed using protein
intrinsic fluorescence. Prior to analysis of the denaturation
curves, the effect of urea on the quaternary structure of Hfq
was determined by rate zonal centrifugation. This allows
discrimination between the unfolded hexameric state (U
and monomeric state (6 U). Figure 4A shows that the
native form of Hfq has an apparent molecular mass of
50 ± 10 kDa, while the unfolded state of Hfq has an
average apparent molecular mass of 12 ± 5 kDa. This
indicates that the unfolded state of Hfq was monomeric in
urea. Urea unfolding of Hfq is accompanied by an overall
increase in the fluorescence intensity, but not by a shift in the
peak maximum (data not shown). This probably results
from a quenching of tyrosine fluorescence in the native state.
As Hfq possesses only three tyrosines (Tyr25, 55 and 83)
and Tyr55 is located in strand 4 [19,20] at the interface
between monomers, exposure of this tyrosine is probably
largely responsible for the increase in fluorescence signal.
The denaturation curves are presented in Fig. 4B. The
simplest model, where hexamer dissociation and protein
unfolding occur in a single step, fits the experimental data
well. We rule out the possibility of a reversible model with
more than two-states because DG° ¼ f [urea] is linear (if
we had a stable intermediate state, we would observe two
transitions and this is not true in our case). Confirmation of
this two-state process is sustained by the observation that
the hexamer dissociation – observed by rate zonal centrifu-
gation – and protein unfolding – exposition of tyrosine or
secondary structure disruption (results not shown) – occur
Fig. 1. Multiple sequence alignment of various bacterial Hfqs. The alignment was produced with T-COFFEE. Amino acids characteristic of Hfq
are indicated in black. Amino acids in light grey are conserved in most Hfqs and located in the b-strands. Acidic amino acids at the end of Hfq
sequences are indicated in white and in dark grey boxes. The alignment clearly indicates that the N-terminal domain is very conserved between
Proteobacteria, Firmicutes, Thermotogales and Aquificales. On the contrary, the C-terminal fragments are variable in length and amino acid
composition. The position of Helix H1 and of the five b-strands are indicated as H1 and E1-E5.Hfq from Photobacterium profundum,
Microbulbifer degradans and Geobacter sulfurreducens are fragments (fr). Firmicutes, Bacillus/clostridium group – Clope, Clostridium perfrin-
gens;Thetn,Thermoanaerobacter tengcongensis. Bacillus/staphylococcus group – Bachd, Bacillus halodurans;Stau,Staphylococcus aureus;
Bacsu, Bacillus subtilis. Proteobacteria c subdivision – Haein, Haemophilus influenzae;Haeso,Haemophilus somnus;Pasmu,Pasteurella
multocida; Phopr, Photobacterium profundum;Vibch,Vibrio cholerae;Xylfa,Xylella fastidiosa;Pseae,Pseudomonas aeruginosa; Pssyr, Pseudo-
monas syringae;Micde,Microbulbifer degradans;Yerpe,Yersinia pestis; Yeren, Yersinia enterocolitica;Salty,Salmonella typhimurium;Shifl,
Shigella flexneri;Ecoli,Escherichia coli;Erwca,Erwinia carotovora; Xanac, Xanthomonas axonopodis;Xancp,Xanthomonas campestris.Pro-
teobacteria d subdivision – Geosu, Geobacter sulfurreducens. Proteobacteria b subdivision – Neima, Neisseria meningitidis;Ralso,Ralstonia
solanacearum. Proteobacteria a subdivision – Caucr, Caulobacter crescentus;Bruab,Brucella abortus;Brume,Brucella melitensis;Azoca,
Azorhizobium caulinodans;Agrtm,Agrobacterium tumefaciens; Rhilo, Rhizobium loti. Thermotogales – Thema, Thermotoga maritima.Aquifi-
cales – Aquae, Aquifex aeolicus.
Ó FEBS 2004 The role of the C-terminal domain on Hfq (Eur. J. Biochem. 271) 1261
simultaneously. For the same reason, we never observed a
hexameric unfolded (U
) form nor oligomeric forms of Hfq
during the dissociation process. The unfolding process
can thus be described by N
Ð 6U. From these curves, the
parameter (midpoint of the transition region)
was measured as 2.9
for Hfq
and 2.4
for Hfq
indicating that the truncated form of Hfq is less stable
than the full-length protein. Based on the analysis of the
transition region, the conformational stability of the
proteins can be calculated [29]: the free energy when
Fig. 2. Image analysis of Hfq oligomers. (A) Average images computed
from 3057 projections for Hfq
(left) and 4833 for Hfq
imposed sixfold symmetry. (B) The corresponding level curves are
represented. A sharper shape is seen for the full-length Hfq projection
compared to the proteolysed; scale bar 2 nm. (C) Significant difference
images (a ¼ 0.05) computed from full-length and proteolysed Hfq
averages. Absolute value of the difference shows the existence of 12
significant regions arranged into two crowns (top). The external crown
corresponds to the positive difference between full-length minus pro-
teolysed Hfq (center left). This difference indicates the absence of
densities in the most external peaks of the full-length oligomer. Pro-
teolysed minus full-length Hfq differences (center right) are significant
at the internal region of the oligomer, as demonstrated by the super-
position of differences in the average image of proteolysed Hfq
(bottom right).
Fig. 3. Affinity of Hfq for polyadenylated rpsO mRNA. The labelled
RNA fragments were incubated with various concentrations of Hfq.
Complexes were separated on native polyacrylamide gels as described
in Materials and methods. Intensities were quantified using a Phos-
Fig. 4. Urea equilibrium unfolding of Hfq. (A) Rate zonal centrifuga-
tion was performed as described above. The final concentration of Hfq
was 5 l
. Samples were subjected to a 15–55% (w/v) saccharose
density gradient and detected with anti-Hfq Igs. Cytochrome c
(12.4 kDa), ovalbumin (44 kDa), bovine serum albumin (67 kDa),
aldolase (158 kDa) and catalase (232 kDa) were used as standards to
calibrate the gradient and are indicated as squares in the gradient.
(B) Denaturation of Hfq monitored by the increase of fluorescence
emission at 303 nm (excitation at 275 nm). Fluorescence emission is
expressed as F
to facilitate comparison. n,Hfq
; s,Hfq
Fluorescence emission was plotted as a function of urea concentration.
From these curves, DG
describing the transition and extrapolated
in water was calculated as 11.5 kCalÆmol
and 9.3 kCalÆmol
for Hfq
and Hfq
1262 V. Arluison et al. (Eur. J. Biochem. 271) Ó FEBS 2004
extrapolated in water ðDG
Þ is estimated to be
11.5 ± 0.3 kCalÆmol
for Hfq
and 9.3 ± 0.25 kCalÆ
for Hfq
. As indicated previously, the His-tag
is located at the C-terminus end of the protein. Thus, the
truncated protein does not carry the His-tag. However,
we do not think that the His-tag is responsible for the
stabilization of the protein because the measured stability
Þ of His-tagged and nonHis-tagged Hfq are almost
As no quantitative data describing the affinity of C-terminal
truncated Hfq for RNA were available, we measured the
equilibrium dissociation constant K
of Hfq
and Hfq
for polyadenylated rpsO RNA. The small change observed
in K
(50 ± 10 p
for Hfq
vs. 120 ± 15 p
for Hfq
indicates that the RNA binding function of the protein is
nearly exclusively located in the N-terminal domain. Our
results agree with previous in vivo studies: indeed, it has
been shown that the 82 amino acid Hfq from Pseudomonas
aeruginosa (92% sequence identity to the N-terminal
domain of Hfq
) and the C-terminally truncated Hfq
(lacking the 27 last amino acids) can functionally replace
full-length Hfq
protein in vivo for phage Qb replication,
rpoS and ompA expression [30]. Our results also agree with
in vitro studies, particularly with the crystal structure of the
77 amino acid S. aureus Hfq bound to a small oligoribo-
nucleotide (AUUUUUG) [19]. This structure indicates that
the first 66 amino acids are sufficient to obtain a complex
between an oligoribonucleotide and Hfq. However, despite
all of these in vitro and in vivo studies, it could not be
excluded that the C-terminal region influences Hfq affinity
for RNA. This is the first direct biochemical evidence that
removal of the C-terminal region does not affect binding of
the polyadenylated rpsO RNA.
We have shown that the presence of the remainder of the
acidic tail results in the thermodynamic stabilization of Hfq
by 1.8 kcalÆmol
. A comparison of the average electron
microscopy images of Hfq
and Hfq
(Fig. 2) also
indicates that proteolysis causes a structural rearrangement
within the protein. The ring of the full-length Hfq is sharper
(not larger) than the ring of the truncated Hfq. This effect in
the truncated form could be explained by a motion of the
segment Ser65–Tyr83 relative to the b5-strand (Ile59–
Pro64). Electron microscopy also indicates that a conform-
ational change probably occurs at the interface between two
consecutive monomers (which involve strands b4ofchainB
and b5 of chain A, Fig. 5). As determined by FTIR, this
rearrangement is coupled to a reduction in the b-content.
On the basis of these observations, we propose that the
C-terminal domain (84–102) protects the interface between
monomers and thus could contribute to the thermodynamic
stabilization of the hexameric Hfq structure. The absence of
this domain results in a reduction of the b-sheet character
within the Sm domain and could perturb the hydrogen
bonding interactions between the interchain strands.
Indeed, it should be noted that FTIR analysis, and
particularly the Amide I vibration that is associated with
the C¼O stretching mode, is a good probe for detecting
variation in the pattern of hydrogen bonding interactions.
The difference D(DG) value found (1.8 kCalÆmol
full-length and truncated Hfq) represents % 20% of the total
free energy of unfolding DG
. We emphasize that the
truncation of Hfq does not break the interface because we
still observe the hexamer in the truncated protein (Fig. 2),
but may destabilize the network of hydrogen bonds.
The crystal structure of the first 71 amino acids of E. coli
Hfq indicates that the C-terminal domain is probably
located at the top of the ring, because the last residues (66–
71) form a short tail pointing towards the a-helices [20].
Taking these results and the electron microscopy data
(Fig. 2) into account, we can assign this region at the
periphery of the ring, to be placed above the Sm-ring, i.e. on
the Ôa-helices faceÕ. This positioning of the C-terminal
protuberance probably protects the hydrophobic part of the
Sm-ring from solvent, particularly at the interfaces between
the subunits, and reinforces our hypothesis of a stabilization
function for this domain. In addition, it should be noted
that the C-terminal domain of Hfq seems to be located at
the same position as that of the Sm-like SmAP3 of the
archaea P. aerophilum [31] and, as in the case of SmAP3, the
possibility of two conformations for the C-terminal domain
could not be excluded.
The multiple sequence alignment of 23 Hfqs using
T-COFFEE [32] presented in Fig. 1 shows that the first
67 amino acids are highly conserved in all species, whereas
the length and sequence composition of the C-terminal
region varies from one species to another. The difference
between the Hfqs results from the insertion of a fragment of
variable length between the first 67 amino acids and the end
of the acidic C-terminal domain. Determining the exact
location of this inserted fragment is not an easy task, as its
length is Hfq-dependent. For example, T-COFFEE fails to
detect a gap for Bacillus subtilis, Pseudomonas aeruginosa
and Brucella melitensis species, because of alignment errors.
Using the phylogenetic tree shown in Fig. 6, based on
evolutionary distances between 16S rRNA and the multiple
Fig. 5. Comparison of the X-ray structure of Hfq
(Protein Data Bank
entry 1HK9 [20]) with the EM projection. (A) Projection of the volume
computed from the X-ray filtered at 18 A
resolution (orange). The
cartoon representation of the X-ray structure has been superimposed.
Each monomer is represented by a different colour. (B) Grey level
curves of the sixfold EM projection. Arrows point to the structural
rearrangement within the protein at the monomers interface. The
shape of this projection is similar to that published by Zhang et al.[11]
with exception of the central region, probably because of a stain
penetration difference. This could also explain that the pore size of the
level curve representation looks higher than that for the X-ray data.
Scale bar 2 nm.
Ó FEBS 2004 The role of the C-terminal domain on Hfq (Eur. J. Biochem. 271) 1263
sequence alignment, we find that long Hfqs are found
exclusively in c-andb proteobacteria. In contrast, the
a proteobacteria, the Firmicutes, the Aquificales and the
Thermotogales lack this inserted fragment. This suggests that
Hfq from c-andb-proteobacteria evolved from the ancestral
sequences by gene fusion. This C-terminal domain may
bring new properties to the protein: for E. coli Hfq, greater
stability. It is also possible that this extension changes the
folding kinetics of Hfq (this is however, beyond the scope
of the present study).
Like Hfq, the C-terminal extension of eukaryotic Sm
proteins was shown to form a protuberance in the Sm
core complex [33]. Several roles were attributed to these
protuberances in Sm. They can, for example, interact
with proteins involved in small nuclear ribonucleoprotein
complex (SnRNP) biogenesis, mediate nuclear localization
or stabilize interactions with RNA [33–36]. In contrast,
our study shows that this extension has rather a structural
role in Hfq. In addition, two major differences between
the C-terminal extension of Sm and Hfq are observed: the
C-terminal domains of Sm proteins are basic and longer
than those of Hfq proteins, which contain mostly acidic
residues. In addition, this extension is highly structured
in some archaeal Sm-like proteins [31], while it is likely
disordered in Hfq. However, this C-terminal extension
may also have a specialized targeting function (either
protein or RNA), in addition to its stability role that we
have invoked.
This work was supported by CNRS (UPR 9073 and 9080) and
University Denis Diderot-Paris 7. We are indebted to J. P. Le Caer
(Ecole polytechnique, Palaiseau, France) and B. Robert (CEA, Saclay,
France) for their help in performing mass spectra and FTIR
experiments. The BL21(DE3) strain transformed with pTE607 plasmid
was kindly provided by T. Elliot. We thank C. Condon for a careful
reading of the manuscript. V. A. was supported by a fellowship from
University Denis Diderot (Paris 7) and M. F. is recipient of a Ph.D.
training grant from MEN.
Fig. 6. Phylogenetic tree of the bacteria based
on the evolutionary distance of the procaryotic
small subunit rRNA phylogenetic. Hfqs har-
bouring a long C-terminal end are underlined.
Hfq of P. profundum, M. degradans and
G. sulfurreducens are fragments and cannot be
classified as long or short Hfq (listing available
at http://rdp.cme.msu.edu/download/
1264 V. Arluison et al. (Eur. J. Biochem. 271) Ó FEBS 2004
1. Franze de Fernandez, M.T., Hayward, W.S. & August, J.T. (1972)
Bacterial proteins required for replication of phage Qb ribonucleic
acid. J. Biol. Chem. 247, 824–821.
2. Muffler, A., Fischer, D. & Hengge-Aronis, R. (1996) The RNA-
binding protein HF-I, known as a host factor for phage Qb RNA
replication, is essential for rpoS translation in E. coli. Genes Dev.
10, 1143–1151.
3. Muffler,A.,Traulsen,D.D.,Fischer,D.,Lange,R.&Hengge-
Aronis, R. (1997) The RNA-binding protein HF-1 plays a global
regulatory role which is largely, but not exclusively, due to its role
in expression of the r
subunit of RNA polymerase in E. coli.
J. Bacteriol. 179, 297–300.
4. Tsui,H C.T.,Leung,H C.,E.&Winkler,M.E.(1994)Char-
acterization of broadly pleiotropic phenotypes caused by an Hfq
insertion mutation in E. coli K-12. Mol. Microbiol. 13, 35–49.
5. Vytvytska, O., Jakobsen, J.S., Balcunate, G., Andersen, J.S.,
Baccarini, M. & von Gabain, A. (1998) Host-factor I, Hfq, binds
to E. coli ompA mRNA in a growth rate-dependent fashion and
regulates its stability. Proc. Natl Acad. Sci. USA 95, 14118–14123.
6. Vytvytska, O., Moll, I., Kaberdin, V.R., von Gabain, A. & Bla
U. (2000) Hfq (HFI) stimulates ompA mRNA decay by interfering
with ribosomes binding. Genes Dev. 14, 1109–1118.
7. Hajnsdorf, E. & Re
gnier, P. (2000) Host factor Hfq of E. coli
stimulates elongation of poly (A) tails by poly (A) polymerase I.
Proc.NatlAcad.Sci.USA97, 1501–1505.
8. Sledjeski, D.D., Whitman, C. & Zhang, A. (2001) Hfq is necessary
for regulation by the untranslated RNA DsrA. J. Bacteriol. 183,
9. Majdalani,N.,Chen,S.,Murrow,J.,StJohn,K.&Gottesman,S.
(2001) Regulation of RpoS by a novel small RNA: the char-
acterization of RprA. Mol. Microbiol. 39, 1382–1394.
10. Majdalani, N., Hernandez, D. & Gottesman, S. (2002) Regulation
and mode of action of the second small RNA activator of RpoS
translation, RprA. Mol. Microbiol. 46, 813–826.
11. Zhang,A.,Wassarman,K.M.,Ortega,J.,Steven,A.C.&Storz,
G. (2002) The Sm-like Hfq protein increases OxyS RNA inter-
action with target mRNAs. Mol. Cell 9, 11–22.
12. Wassarman, K.M., Repoila, F., Rosenow, C., Storz, G. & Got-
tesman, S. (2001) Identification of novel small RNAs using com-
parative genomics and microarrays. Genes Dev. 15, 1637–1651.
13. Masse, E., Majdalani, N. & Gottesman, S. (2003) Regulatory roles
for small RNA in bacteria. Cur. Opinion Microbiol. 6, 120–124.
14. Moll, I., Leitsch, D., Steinhauser, T. & Blasi, U. (2003) RNA
chaperone activity of the Sm-like Hfq Protein. EMBO Reports 4,
15. Moller, T., Franch, T., Hojrup, P., Keene, D.R., Bachinger, H.P.,
Brennan, R.G. & Valentin-Hansen, P. (2002) Hfq. A bacterial
Sm-like protein that mediates RNA–RNA interaction. Mol. Cell
9, 23–30.
16. Arluison, V., Derreumaux, P., Allemand, F., Folichon, M.,
Hajnsdorf, E. & Regnier, P. (2002) Structural modelling of the
Sm-like protein Hfq from E. coli. J. Mol. Biol. 320, 705–712.
17. Seraphin, B. (1995) Sm and Sm-like proteins belong to a large
family: identification of proteins of the U6 as well as the U1, U2,
U4 and U5 snRNPs. EMBO J. 14, 2089–2098.
18. He, W. & Parker, R. (2000) Functions of Lsm proteins in mRNA
degradation and splicing. Curr. Opinion Cell Biol. 12, 346–350.
19. Schumacher, M.A., Pearson, R.F., Moller, T., Valentin-Hansen,
P. & Brennan, R.G. (2002) Structures of the pleiotropic transla-
tional regulator Hfq and an Hfq- RNA complex: a bacterial Sm-
like protein. EMBO J. 21, 3546–3556.
20. Sauter, C., Basquin, J. & Suck, D. (2003) Sm-like proteins in
Eubacteria: the crystal structure of the Hfq protein from E. coli.
Nucleic Acids Res. 31, 4091–4098.
21. Bradford, M. (1976) A rapid and sensitive method for the quan-
tification of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248–254.
22. Schagger, H. & von Jagow, G. (1987) Tricine-sodium dodecyl
sulfate-polyacrylamide gel electrophoresis for the separation of
proteins in the range from 1 to 100 kDa. Anal. Biochem. 166,
23. Marabini,R.,Masegosa,I.M.,SanMartin,C.,Marco,S.,Fer-
nandez, J.J., de la Fraga, L.G., Vaquerizo, C. & Carazo, J.M.
(1996) Xmipp: An image processing package for electron micro-
scopy. J. Struct. Biol. 116, 237–240.
24. Marabini, R. & Carazo, J.M. (1994) Pattern recognition and
classification of images of biological macromolecules using artifi-
cial neural networks. Biophys. J. 66, 1804–1814.
25. Unser, M., Trus, B.L. & Steven, A.C. (1987) A new resolution
criterion based on spectral signal-to-noise ratios. Ultramicroscopy
23, 39–51.
26. Folichon, M., Arluison, V., Pellegrini, O., Huntzinger, E., Reg-
nier, P. & Hajnsdorf, E. (2003) The poly (A) binding protein Hfq
protects RNA from RNase E and exoribonucleolytic degradation.
Nucleic Acids Res. 31, 7302–7310.
27. Zhang, A., Altuvia, S., Tiwari, A., Argaman, L., Hengge-Aronis,
R. & Storz, G. (1998) The oxyS regulatory RNA represses rpoS
translation and binds the Hfq (HF-1) protein. EMBO J. 17,
28. Byler, D.M. & Susi, H. (1986) Examination of the secondary
structure of proteins by deconvolved FTIR spectra. Biopolymers
25, 469–487.
29. Pace, N.R. (1986) Determination and analysis of urea and
guanidine hydrochloride denaturation curves. Methods Enzymol.
131, 266–280.
30. Sonnleitner, E., Moll, I. & Blasi, U. (2002) Functional replacement
of the E. coli Hfq gene by the homologue of Pseudomonas aeru-
ginosa. Microbiology 148, 883–891.
31. Mura, C., Phillips, M., Kozhukhovsky, A. & Eisenberg, D. (2003)
Structure and assembly of an augmented Sm-like archaeal protein
14-mer. Proc. Natl Acad. Sci. USA 100, 4539–4544.
32. Notredame, C., Higgins, D.G. & Heringa, J. (2000) T-Coffee: a
novel method for fast and accurate multiple sequence alignment.
J. Mol. Biol. 302, 205–217.
33. Bordonne, R. (2000) Functional characterization of nuclear
localization signals in yeast Sm proteins. Mol. Cell Biol. 20, 7943–
34. Mouaikel, J., Verheggen, C., Bertrand, E., Tazi, J. & Bordonne, R.
(2002) Hypermethylation of the cap structure of both yeast
snRNAs and snoRNAs requires a conserved methyltransferase
that is localized to the nucleolus. Mol. Cell 9, 891–901.
35. Brahms, H., Meheus, L., de Brabandere, V., Fischer, U. &
Luhrmann, R. (2001) Symmetrical dimethylation of arginine
residues in spliceosomal Sm protein B/B’ and the Sm-like pro-
tein LSm4, and their interaction with the SMN protein. RNA 7,
36. Zhang, D., Abovich, N. & Rosbash, M. (2001) A biochemical
function for the Sm complex. Mol. Cell 7, 319–329.
Ó FEBS 2004 The role of the C-terminal domain on Hfq (Eur. J. Biochem. 271) 1265

Tài liệu bạn tìm kiếm đã sẵn sàng tải về

Tải bản đầy đủ ngay