The C-terminal domain of Escherichia coli Hfq increases the stabilityof the hexamer Ve´ronique Arluison1, Marc Folichon1, Sergio Marco2, Philippe Derreumaux3, Olivier Pellegrini1, Je´roˆ
Trang 1The C-terminal domain of Escherichia coli Hfq increases the stability
of the hexamer
Ve´ronique Arluison1, Marc Folichon1, Sergio Marco2, Philippe Derreumaux3, Olivier Pellegrini1,
Je´roˆme Seguin4, Eliane Hajnsdorf1and Philippe Regnier1
1
Institut de Biologie Physico-Chimique CNRS UPR 9073 conventionne´e avec l’universite´ Paris 7, Paris, France;2Institut Curie CNRS UMR 168, Paris, France;3Institut de Biologie Physico-Chimique CNRS UPR 9080, Paris, France;4Service 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 coliHfq 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 (Kdof 120 pMvs 50 pMfor
full-length Hfq) This result shows that the functional core of
E coliHfq 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)1more 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 rS subunit of RNA
poly-merase and, as a consequence, influences the expression of
many stationary phase genes whose transcription depends
on rS[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 ec , E coli Hfq;
Hfq f , Hfq full-length; Hfq Nter , Hfq lacking the 19 last amino acid;
sRNAs, small RNAs.
(Received 19 December 2003, revised 2 February 2004,
accepted 6 February 2004)
Trang 2the 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 mM Tris/HCl, pH 7.8, 0.5
MNaCl, 10% (v/v) glycerol and 0.1% (v/v) Triton X-100
at 4C 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 mM The resulting
suspension was applied to a 1 mL Ni2+–nitrilotriacetic
acid column (Qiagen) The resin was then washed
sequentially with 15 column volumes of: (a) 20 mM
Tris/HCl, pH 7.8 buffer containing 0.3M NaCl and
20 mM imidazole and (b) 50 mM sodium phosphate,
pH 6.0 buffer containing 0.3MNaCl, and Hfq was finally
eluted with a buffer containing 50 mMsodium phosphate,
pH 6.0, 0.3MNaCl and 250 mMimidazole The fractions
containing Hfq were analysed by SDS/PAGE, pooled
and heated to 80C for 15 min Insoluble material was
removed by centrifugation and the supernatant was
dialysed in buffer containing 50 mM Tris/HCl, pH 7.5,
1 mMEDTA, 50 mMNH4Cl, 5% (v/v) glycerol and 0.1%
(v/v) Triton X-100 The protein was stored at 4C
Protein concentrations were determined by measuring the
absorption at 280 nm (e280 at 1 mgÆmL)1¼ 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 20C in 50 m
Tris/HCl, pH 8 containing 100 mM NaCl Chymotrypsin (1 ng) was used for 1 lg of Hfq (concentration
1 mgÆmL)1, 200 lL) Aliquots of 20 lL were withdrawn
at different times and the reaction was stopped by adding the universal protease inhibitor a2-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 spacer gel The N-terminal fragment (HfqNter) generated by chymotrypsin ( 9 kDa) was purified by extensive dialysis (molecular mass cut-off 3500 Da) against 10 mM Tris/HCl, pH 8, buffer containing 80 mMNaCl, 1% (v/v) glycerol, 0.01% (w/v) dodecyl-b-D-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 H2O/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 (Hfqf) and proteolysed Hfq (HfqNter) 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 Hfqf, and 4833 from HfqNter, were windowed and aligned by usingX-MIPPsoftware [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 SSNRmethod [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 X-MIPP software with a significance level a¼ 0.05
Gel-shift assay Labelled RNA fragments (2.5 pM) were incubated with Hfq for 20 min at 37C in 50 lL of 10 mM Tris/HCl, pH 8 buffer containing 80 mMNaCl, 1% (v/v) glycerol, 0.01% (w/v) dodecyl-b-D-maltoside The RNA used consisted of rpsOmRNA with a 18 nucleotide poly(A) tail [7] The RNA was titrated with an excess of Hfq, its concentration ranging from 15 pM to 10 nM[26] Complexes were separated on native PAGE as described in Zhang et al [27]
Trang 3Infrared spectra
Attenuated total reflectance (ATR)-FTIR spectra of Hfq
[200 lM, previously dialysed overnight against 10 mMTris/
HCl, pH 8 containing 80 mM NaCl and 0.01% (w/v)
dodecyl-b-D-maltoside] were measured with a Bruker vector
22 spectrophotometer equipped with a 45n diamond ATR
attachment at 20C The spectra are the average of 125
scans Spectra were corrected for the linear dependence of
the absorption measured by ATRon 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, DGu, fits
to equation:
DGu ¼ DGH2 O
u m½urea;
where, DGH2 O
u 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 20C, overnight, at 40 000 r.p.m., in the
70 Ti rotor of a Beckman LE-80 ultracentrifuge Hfq was
denatured in a 50 mM Tris/HCl pH 8 buffer containing
100 mM NaCl and 6M urea prior to centrifugation
Samples (100 lL) of 10 lM Hfq were applied on the top
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)
Results
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 HfqEc)
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 Hfqfand HfqNterwas 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 (a¼ 0.05) showed that Hfqfand HfqNter 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 b4 of chain B and the 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 FTIRspectra 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 FTIRbeing 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 FTIRspectrum of the C-terminal peptide is strongly dominated by a broad band
at 1641 cm)1, 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 and
Trang 4HfqNter The corresponding equilibrium dissociation
con-stants (Kd) were found to be 50 ± 10 pMand 120 ± 15 pM
for Hfq and HfqNter, respectively The minor increase in
Kd 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 (U6)
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 ProPro-teobacteria b subdivision – Neima, Neisseria meningitidis; R also, 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.
Trang 5simultaneously For the same reason, we never observed a
hexameric unfolded (U6) form nor oligomeric forms of Hfq
during the dissociation process The unfolding process
can thus be described by N6Ð 6U From these curves, the [urea]1/2 parameter (midpoint of the transition region) was measured as 2.9M for Hfqf and 2.4M for HfqNter, 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 f (left) and 4833 for Hfq Nter with
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-phorImager.
Fig 4 Urea equilibrium unfolding of Hfq (A) Rate zonal centrifuga-tion was performed as described above The final concentracentrifuga-tion of Hfq was 5 l M 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 app to facilitate comparison n, Hfq f ; s, Hfq Nter Fluorescence emission was plotted as a function of urea concentration From these curves, DGH2 O
u describing the transition and extrapolated
in water was calculated as 11.5 kCalÆmol)1and 9.3 kCalÆmol)1for Hfq and Hfq Nter
Trang 6extrapolated in water ðDGH 2 O
u Þ is estimated to be 11.5 ± 0.3 kCalÆmol)1 for Hfqf and 9.3 ± 0.25 kCalÆ
mol)1 for HfqNter 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
ðDGH2O
u Þ of His-tagged and nonHis-tagged Hfq are almost
identical
Discussion
As no quantitative data describing the affinity of C-terminal
truncated Hfq for RNA were available, we measured the
equilibrium dissociation constant Kdof Hfqfand HfqNter
for polyadenylated rpsO RNA The small change observed
in Kd(50 ± 10 pMfor Hfqfvs 120 ± 15 pMfor HfqNter)
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 HfqEc) and the C-terminally truncated HfqEc
(lacking the 27 last amino acids) can functionally replace
full-length HfqEc protein in vivo for phage Qb replication,
rpoSand ompA expression [30] Our results also agree with
in vitrostudies, 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)1 A comparison of the average electron
microscopy images of HfqNter and Hfqf (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 b4 of chain B
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 FTIRanalysis, 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)1between
full-length and truncated Hfq) represents 20% of the total free energy of unfolding DGH2 O
u 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 a-helices face 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 Ec (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.
Trang 7sequence alignment, we find that long Hfqs are found
exclusively in c- and b proteobacteria In contrast, the
a proteobacteria, the Firmicutes, the Aquificales and the
Thermotogales lack this inserted fragment This suggests that
Hfq from c- and b-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
Acknowledgements
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/ SSU_rRNA/SSU_Prok.phylo).
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