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J. Biol. Chem., Vol. 275, Issue 40, 31428-31437, October 6, 2000
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From the
Received for publication, April 5, 2000, and in revised form, May 11, 2000
Oligomerization of viral envelope proteins is
essential to control virus assembly and fusion. The transmembrane
domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2
have been shown to play multiple functions during the biogenesis of
E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the
assembly of E1E2 heterodimer. Alanine insertion within the center of
the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1
dramatically reduced heterodimerization, demonstrating the essential
role played by these domains in the assembly of hepatitis C virus
envelope glycoproteins. To better understand the alanine scanning data
obtained for the TMD of E1 which contains GXXXG motifs, we
analyzed by circular dichroism and nuclear magnetic resonance the
three-dimensional structure of the E1-(350-370) peptide
encompassing the N-terminal sequence of the TMD of E1 involved in
heterodimerization. Alanine scanning results and the three-dimensional
molecular model we obtained provide the first framework for a molecular
level understanding of the mechanism of hepatitis C virus envelope
glycoprotein heterodimerization.
After their synthesis and integration into the membrane, a large
number of membrane proteins associate and form homo- or
hetero-oligomeric complexes with new functions. To ensure specific
assembly, these proteins must present complementary recognition regions
to each other. These interacting regions may be located on the
ectodomains and/or the transmembrane sequences. The importance of
noncovalent interactions between transmembrane HCV is the causal agent of hepatitis C which is a major health problem
worldwide. It is a positive stranded RNA virus and is a member of the
hepacivirus genus in the family Flaviviridae (5). Its genome
encodes a single polyprotein of about 3000 amino acid residues that is
co- and post-translationally cleaved to generate at least 10 polypeptides (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (6).
The lack of a cell culture system supporting efficient HCV replication
and particle assembly have hampered the characterization of the
envelope proteins present on the virion. However, indirect evidences,
like viral neutralization by antibodies, support the idea that HCV
envelope glycoproteins are present on the surface of the virion (7).
Our current understanding of the assembly of HCV envelope glycoproteins
is based on cell culture transient-expression assays with viral or
nonviral expression vectors. E1 and E2 are obtained after cleavage of
the polyprotein by host signal peptidase(s) (8). They are heavily
modified by N-linked glycosylation and are transmembrane
proteins with a large N-terminal ectodomain and a C-terminal
hydrophobic anchor (9). E1 and E2 glycoproteins have been shown to
assemble as a noncovalent heterodimer (10). However, this process is
not efficient, and misfolded heterogeneous disulfide-linked aggregates of E1 and E2 are also produced, at least when using heterologous expression systems (9). Coexpression of E1 and E2 has been shown to be
necessary to ensure correct folding of E1 (11). The two glycoproteins
likely play separate functions in HCV entry. Indeed, E2 might be
involved in receptor binding (12, 13), whereas E1 has been proposed to
be the fusion protein (14).
The fact that the envelope proteins of HCV are translated from a single
coding region implies that internal signal peptides must be used. The
signal sequences of E1 and E2 are present at the C terminus of the
immature form of the capsid protein and in the second half of the TMD
of E1, respectively (8). In addition, a hydrophobic sequence present in
the second half of the TMD of E2 is the signal sequence for a
polypeptide called p7. Recently, we showed that the TMDs of E1 and E2
play a major role in the subcellular localization of E1·E2
complex (15-17). The TMDs of E1 and E2 have also been suggested to
play a major role in the assembly of the heterodimer. Indeed, deletion
of the C-terminal hydrophobic sequence of E2 (11, 18), or its
replacement by the membrane anchor of CD4 or a
glycosylphosphatidylinositol moiety, have been shown to abolish the
formation of E1·E2 complexes (15).
The multiple functions played by the TMDs of E1 and E2 glycoproteins
are supposed to be essential for the formation of the viral envelope,
and most likely impose limitations on the amino acid variability of
these domains (3). These TMDs are composed of two stretches of
hydrophobic residues separated by a short segment containing at least
one fully conserved charged residue (Fig. 1). Replacement of these
charged residues by alanine has been shown to alter all the functions
of the TMDs of E1 and E2 (3), indicating that charged residues present
within these domains are crucial for their multifunctionality.
In this report, we studied the role played by the TMDs of E1 and E2 in
heterodimerization. We used alanine scanning insertion mutagenesis (19,
20) in these domains to examine their role in the assembly of the
heterodimer. This technique has been shown to be a powerful method to
detect dimerization of transmembrane Cell Culture--
The HepG2, CV-1, and 143B (thymidine
kinase-deficient) cell lines were obtained from the American Type
Culture Collection, Rockville, MD. Cell monolayers were grown in
Dulbecco's modified essential medium (Life Technologies) supplemented
with 10% fetal bovine serum.
Plasmid Constructs--
Plasmids expressing wild type and
mutated E1E2 polyproteins (signal sequence of E1, and the sequences of
E1 and E2) were constructed by standard methods (21). Briefly, DNA
sequences of HCV proteins were polymerase chain reaction
amplified and introduced into plasmid pTM1 (22). HCV sequences were
amplified with the appropriate oligonucleotides from H strain clones
(23, 24). Site-directed mutagenesis was performed by enzymatic inverse
polymerase chain reaction as described by Stemmer and Morris (25). A
plasmid expressing the chimeric protein CD4-E1A358' was constructed as described (16). This plasmid contains the sequence of the signal peptide of CD4, followed by the sequence of the ectodomain of CD4 in
fusion with the sequence encoding the C-terminal 37 amino acids of E1
with an alanine inserted at position 358'.
Generation and Growth of Viruses--
Vaccinia virus
recombinants were generated by homologous recombination essentially as
described (26) and plaque purified twice in 143B cells under
bromodeoxyuridine selection (50 µg/ml). Stocks of vTF7-3 (a vaccinia
virus recombinant expressing the T7 DNA-dependent RNA
polymerase) (27), the wild type vaccinia virus strain, Copenhagen, its
thermosensitive derivative ts7 (28), and vaccinia virus recombinants
expressing HCV proteins or mutated proteins were grown and titrated on
CV-1 monolayers.
Antibodies--
Monoclonal antibodies (mAbs) H53 (anti-E2;
(15)), A4 (anti-E1 (29)), and OKT4 (anti-CD4 (30)) were produced
in vitro by using a MiniPerm apparatus (Heraeus) as
recommended by the manufacturer.
Metabolic Labeling and Immunoprecipitation--
Cells were
infected with the appropriate vaccinia virus recombinants and
metabolically labeled with 35S-Protein Labeling Mix
(3.7 × 106 Bq/ml; NEN Life Science Products) as
described previously (29). Cells were lysed with 0.5% Triton X-100 in
Tris-buffered saline (TBS) (50 mM Tris-Cl (pH 7.5), 150 mM NaCl). Immunoprecipitations were carried out as
described previously (31). For in vivo labeling of glycan
moieties, HepG2 cells were infected with the appropriate vaccinia virus
recombinants and pulse-labeled for 30 min with [2-3H]mannose (3.7 × 106 Bq/ml;
Amersham Pharmacia Biotech) in Analysis of Oligosaccharide Material--
Immunoprecipitated
[2-3H]mannose-labeled proteins were digested
overnight at room temperature with 0.2 mg of
N-tosyl-L-phenylalanine chloromethyl
ketone-treated trypsin in 0.1 M ammonium bicarbonate (pH
7.9). Trypsin-treated proteins were boiled for 10 min to inactivate the
trypsin, and the peptides were lyophilized and dissolved in 20 mM sodium phosphate (pH 7.5) containing 50 mM
EDTA and 0.2 mg of NaN3/ml in 50% glycerol. The peptides
were incubated overnight at 37 °C in the presence of peptide
N-glycanase F (0.5 units; New England Biolabs). Size
analysis of the glycan moieties was achieved by high pressure liquid
chromatography (HPLC) on an amino-derivatized column ASAHIPAK
NH2P-50 (250 by 4.6 mm) (Asahi, Kawasaki-ku, Japan) with a
solvent system of acetonitrile/water from 70:30 (v/v) to 50:50 (v/v) at
a flow rate of 1 ml/min over 80 min. Oligomannosides were identified as
described previously (32) by their retention time. Separation of
labeled oligosaccharides was monitored by continuous flow detection of
radioactivity with a Flo-One Peptide Synthesis and Samples Preparation--
E1-(350-370)
synthetic peptide was purchased from G. Blomberg, School of Medical
Sciences, University of Bristol (United Kingdom). The numbering of this
synthetic peptide (denoted E1-(350-370)) refers to the genotype 1a and
its sequence (GAHWGVLAGIAYFSMVGNWAK-NH2) is identical to
that of an infectious HCV cDNA clone recently published (33) (EMBL
access number: AF009606). The synthesis was performed using N-tBoc
chemistry and the C terminus was blocked by an amide group. Purity of
the peptide was assessed by electrospray mass spectroscopy (molecular
mass 2234 Da) and appeared to be higher than 90%. Moreover,
only a minor spin system due to the impurity of the sample was observed
in the NMR spectra. The peptide was rather insoluble in water but
readily soluble in the presence of trifluoroethanol (TFE). Micellar
solutions were prepared by adding concentrated aqueous solutions of
lysophosphatidylcholine (LPC) or SDS to a solution of E1-(350-370)
dissolved in 90% TFE. Some TFE was eventually added to yield a clear
and homogeneous mixture. After freezing and lyophilization to remove
any trace of TFE, the samples were solubilized in 10 mM
phosphate (pH 7.4). Micellar solutions were routinely checked for
background absorbance, from which it was noted that light scattering
was insignificant at the SDS and LPC concentrations used. Peptide
concentrations were determined by amino acid analysis.
CD Measurements--
CD spectra were recorded on a Jobin-Yvon
CD6 spectrometer calibrated with ammonium
d10-camphorsulfonate. Routinely, measurements were done at 298 K in 0.1-cm path length quartz cuvettes (Hellma) with
peptide concentrations ranging from 30 to 50 µM. Spectra were recorded in the 190-250 nm wavelength range with 0.2-nm
increments and 2-s integration time. The baseline-corrected spectra
were smoothed by using a third-order least squared polynomial fit. Assuming that the residue molar ellipticity at 222 nm is exclusively due to 1H NMR Spectroscopy--
Lyophilized peptide was
dissolved in an aqueous solution containing 50%
TFE-d2
(2,2,2-trifluoroethyl-1,1-d2 alcohol >99%
isotopic enrichment). The final peptide concentration was 4 mM and the pH measured was 5.7 (uncorrected). Sodium
2,2-dimethyl-2-silapentane-5-sulfonate was added as an internal
reference. All NMR experiments were recorded at 500 MHz on a Varian
Unity-plus spectrometer. Spectra were acquired non-spinning
at temperatures of 293 and 303 K. Two-dimensional homonuclear
1H experiments (DQF-COSY, Clean-TOCSY, and NOESY) were
performed according to the conventional pulse sequences. Water
suppression was carried out using selective, low power irradiation
during the 1.5-s relaxation delay and during the mixing time in NOESY experiments. Routinely, the spectra were recorded with 6000 Hz spectral
width in both dimensions and data sets collected as 512 and 2048 points
in t1 and t2 dimensions,
respectively, with 32 or 64 scans per increment. Data collection and
processing were carried out as detailed previously (36, 37). The
resonances of protons were ascribed by the conventional assignment
method (38).
NMR-derived Constraints and Structure Calculations--
NOE
intensities used as input for structure calculations were obtained from
the NOESY spectrum recorded with a 300-ms mixing time and checked for
spin diffusion on spectra recorded at lower mixing times (50-150 ms).
Spectra obtained at 303 K were also used to estimate NOE intensities
for cross-peaks unresolved at 293 K. NOEs were partitioned into four
categories of intensities that were converted into distances ranging
from a common lower limit of 1.8 Å to upper limits of 2.8, 3.9, 5.0, and 6.0 Å, respectively. The cross-peak intensity of the
H Identification of TMD Segments Involved in Heterodimerization
To confirm the role played by the TMDs of HCV envelope
glycoproteins in heterodimerization, we used alanine scanning insertion mutagenesis, a technique which has been shown to disrupt helix-helix interaction in a membrane environment (19, 20). The TMDs of E1 and E2
are present at the C terminus of these proteins (Fig. 1). The N-terminal amino acids of these
domains have not been precisely determined but it has been predicted
that they could start at position 353 and 718 for E1 and E2,
respectively (16, 42). A series of mutated proteins were obtained by
introducing a single alanine residue in the TMD of E1 or E2 (Fig. 1).
Since HCV envelope glycoproteins are produced after cleavage of a
polyprotein, these mutations were introduced in the context of an E1E2
polyprotein. Mutated E1E2 polyproteins were expressed in HepG2 cells by
using the vaccinia virus expression system. The ability of the mutants to form a noncovalent E1E2 complex was analyzed by immunoprecipitation with a conformation-sensitive E2-specific mAb (H53) which has been
shown to specifically precipitate the native E1·E2 complex (15, 17).
Cells expressing mutated E1E2 polyproteins were pulse-labeled with
35S-Protein Labeling Mix for 10 min and chased for 4 h. These conditions have been shown to be appropriate to detect the
peak of heterodimer formation (10). To evaluate the level of expression
of E1, a control immunoprecipitation with a conformation-insensitive
anti-E1 mAb (A4) was performed. E1 expression was found to be constant whatever the alanine insertions (Fig.
2A). Since mAb H53 is
E2-specific, and because E2 can fold independently of E1 (11), the
amount of E1 co-precipitated by mAb H53 is a good indicator of the
assembly of the noncovalent heterodimer. To evaluate the percentage of heterodimerization, E1/E2 ratios were measured for each mutant and
compared with the ratio obtained with wild type proteins. As shown in
Fig. 2, a severe disruption of heterodimerization was observed when
alanine residues were introduced at positions Ala355',
Ala358', Ala361', or Ala369' of the
TMD of E1. Similar results were observed for E2 mutants Ala727' and Ala730' (Fig.
3); however, the effects of alanine
insertion were less dramatic than for E1 mutants. Indeed, for E1
mutants Ala355', Ala358', Ala361',
and Ala369', heterodimerization was reduced to less than
10% of the wild type level (Fig. 2B), whereas about 35% of
E1·E2 complex was still detected for the most disruptive mutations in
E2 (Fig. 3; Ala727', Ala730'). It is noteworthy
that the intensity of E2 precipitated by mAb H53 was lower for some of
the E1 mutants impaired in heterodimerization (Fig. 2A). It
is likely that in the absence of heterodimerization, E1 which does not
fold properly in such conditions (see below) interferes with the
folding of E2 by forming disulfide-linked aggregated complexes. Since
insertion of an alanine close to the predicted N-terminal amino acid of
the TMD of E1 impaired E1E2 assembly (Fig. 2, Ala355'), two
additional mutations were introduced outside of this domain (positions
346' and 352', see Fig. 1). Insertion at position 346' had only a
moderate effect on heterodimerization, whereas E1E2 assembly was
reduced to approximately 30% for the Ala352' mutant (Fig.
2), suggesting that a segment involved in E1E2 interaction could extend
outside of the TMD of E1. Alternatively, the N terminus of the TMD of
E1 might be located upstream of its predicted position. Interestingly,
mutations at positions 364' and 367' of E1 were less disruptive than in
surrounding positions (Fig. 2B). These data suggest that two
distinct segments involved in heterodimerization are present in E1. It
is noteworthy that the second of these segments is located close to the
charged residue which has been reported to be important for the
multifunctionality of the TMD of E1 (3). Similarly, the segment
identified in the TMD of E2 might include the two charged residues
involved in the multifunctionality of this domain (Fig. 1).
Interestingly, no change in the efficiency of cleavage of E1E2
polyprotein was observed for E1 mutants (data not shown), indicating
that the signal sequence function located in the TMD of E1 is not
affected by an alanine insertion in this domain.
Assembly of the native E1·E2 complex is not efficient. Indeed,
misfolded disulfide-linked aggregates are abundant in cells expressing
these proteins (9). We were interested to know whether the mutations
introduced in the TMD of E1 or E2 would also affect the formation of
these aggregates. For this purpose, we used a mAb that we recently
described as specific of E1E2 aggregates (43), and we analyzed the
formation of E1E2 aggregates in pulse-chase experiments. Similar levels
of aggregates were observed for all the alanine mutants (data not
shown), indicating that only noncovalent complex formation is impaired
by the insertion of an alanine in segments critical for assembly.
Altogether, our data indicate that segments located close to the
charged residue(s) of the TMDs of E1 and E2 are directly involved in
heterodimerization. In addition, a second segment playing a role in
E1E2 assembly is present near residue 358 of the TMD of E1.
Mutants Impaired in Heterodimerization Retain Their ER Retention
Function
Since the TMDs of E1 and E2 are ER retention signals (15, 16), we
wanted to determine whether this function would be maintained despite
defective assembly of some mutants. To answer this question, we
determined the subcellular localization of some of the mutants showing
the most disruptive effect on assembly (Ala358' and
Ala727') by analyzing the type of glycans associated with
these proteins. We have shown previously that the type of glycans
associated with HCV envelope glycoproteins, or chimeras having the TMD
of E1 or E2, is a good indicator of their subcellular localization (16, 17). To analyze the glycans associated with an E2 mutant, we used a
vaccinia virus recombinant expressing E2-Ala727' alone.
Since E1 does not fold properly in the absence of E2 (11), we analyzed
the subcellular localization of a chimeric protein
CD4-E1-Ala358', comprising the ectodomain of CD4 fused to
the TMD of E1-Ala358', and expressed by using a vaccinia
vector. Such a strategy has been used previously to demonstrate that
the TMD of E1 is an ER retention signal (16).
To evaluate the level of processing of the glycans associated with
E2-Ala727' or CD4-E1-Ala358', HepG2 cells
expressing these proteins were pulse-labeled with [2-3H]mannose, chased for 4 h, and used for
immunoprecipitation with an anti-E2 (H53) or anti-CD4 (OKT4) mAb.
Glycans associated with these proteins were removed by peptide
N-glycanase F treatment and analyzed by HPLC. Such an
analysis demonstrated the presence of three species: Man9,
Man8, and Man7-GlcNAc2, respectively (Fig.
4). These glycans were similar to those
observed for CD4-E1 and -E2 (16, 17) and typical of ER-retained
glycoproteins, indicating that the alanine residues introduced at
position 358' of the TMD of E1 and at position 727' of E2 do not modify
the subcellular localization of CD4-E1 and -E2, respectively.
The Transmembrane Domains of Hepatitis C Virus Envelope
Glycoproteins E1 and E2 Play a Major Role in Heterodimerization*
,,
, and§§
CNRS-UMR8526, IBL/Institut Pasteur de Lille,
59021 Lille Cedex, France, the § CNRS-UMR 5086, IBCP, 69367 Lyon Cedex 07, France, the ¶ CNRS-UMR 8576/USTL, 59655 Villeneuve
d'Ascq Cedex, France, and the ** CEA/CNRS-URA 2096, 91191 Gif/Yvette Cedex, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices in
oligomerization of membrane proteins is becoming increasingly apparent
(1, 2). However, interactions of transmembrane -helices have only been well characterized for a restricted number of proteins and mainly
in the context of homo-oligomerization. Recently, we have shown that
the transmembrane domains
(TMDs)1 of hepatitis C virus
(HCV) E1 and E2 envelope glycoproteins are required in
heterodimerization (3). Therefore, these proteins constitute an
attractive model to study hetero-oligomerization of transmembrane
domains. In addition, like other viral envelope proteins, HCV
glycoproteins are supposed to play a crucial role in viral entry by
binding to a receptor present on the host cell and inducing fusion
between the viral envelope and a membrane of the host cell (4). To
ensure that fusion does not occur during protein synthesis and folding,
nor during budding of the viral particle, this function has to be
tightly controlled. It is well known that oligomerization plays a
crucial role in regulating the fusogenic function of such proteins (4).
Studying the early steps of oligomerization of HCV envelope
glycoproteins is essential to understand how the virus controls its
fusion activity.
-helices. Indeed, insertion of
a single amino acid into a transmembrane helix displaces the residues
on the N-terminal side of the insertion by 110° relative to those on
the C-terminal side of the insertion, disrupting a helix-helix packing
interface involving residues on both sides of the insertion. Two
distinct segments of the TMD of E1 and one of the TMD of E2 were very
sensitive to alanine insertion, demonstrating the essential role played
by these domains in the assembly of HCV envelope glycoproteins. Neither
ER retention nor signal sequence cleavage were affected by these
mutations, indicating that assembly can be dissociated from other
functions played by the TMDs of HCV envelope glycoproteins. In
complement to the alanine scanning data obtained for the N-terminal
part of TMD of E1 which contains GXXXG motifs, we
used circular dichroism (CD) and nuclear magnetic resonance (NMR) to
analyze, in the presence of various membrane mimetic solvents
(detergents and trifluoroethanol/water mixtures), the
three-dimensional structure of an E1 peptide encompassing the sequence
involved in heterodimerization (E1-(350-370) segment). Alanine
scanning results and the three-dimensional molecular model we obtained
provide the first framework for a molecular level understanding of the
mechanism of E1E2 heterodimerization. To our knowledge, this is the
first report showing that oligomerization of viral envelope proteins
can be controlled by transmembrane sequences.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimum essential medium containing
0.5 mM glucose and 10% dialyzed fetal bovine serum. After
4 h of chase, cells were lysed in TBS, 0.5% Triton X-100, and the lysates were used for immunoprecipitation.
detector (Packard).
-helix (34), the -helical content was estimated using the
following equation: % helix = 100[
]obs
/[]H, where []obs is the observed mean residue ellipticity and
[]H is the maximum mean residue ellipticity for a
helix of finite length. []H was calculated
using the empirical equation for helix-length dependence proposed by
Chen et al. (35) []H = []H
(fH 
ik/n), where
[]H is the maximum mean residue
ellipticity for a helix of infinite length (39,500 for 
= 222 nm), fH is the fraction of helix, i
is the number of helical segments (1 in this case), k is a
wavelength dependent constant (2.57 for = 222 nm), and
n is the number of residues.
-H
protons of Phe362
was used as a distance reference (2.45 Å). Protons without
stereospecific assignments were treated as pseudoatoms, and correction
factors were added to the upper and lower distance constraints (38). NOEs back-calculations were performed from calculated structures (see
below) by using the standard procedure of X-PLOR 3.1 program (39).
Three-dimensional structures were generated from NOE distances with
X-PLOR 3.1, using the standard force fields and default parameter sets,
except some minor modifications (36) to increase the duration of the
molecular dynamic simulations and the number of energy minimization
steps. A group of 50 structures was calculated to widely sample the
conformational space and the structures were selected on the basis of
low energy and NOE violations. The structures were compared by pairwise
RMSD for the backbone atom coordinates (N, C, and C'). Local
analogies were analyzed by calculating the local RMSD of a tripeptide
window slided along the sequence. Statistical analysis, superimposition
of structures, three-dimensional graphic displays, and manipulations
were achieved by using RASMOL 2.5 software (40). The secondary
structure elements and Ramachandran plots were analyzed according to
the Kabsch-Sander definition rules, as incorporated in the program
PROCHEK-NMR (41).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 1.
A, schematic representation of HCV
polyprotein in the region comprising the envelope glycoproteins. Signal
sequences and TMDs are indicated by solid boxes. The
arrows indicate the sites which are cleaved by a signal
peptidase. The amino acid positions of the N terminus of E1, E2, and p7
are shown. sp is the signal sequence of E1. B,
positions of the insertion mutations in the C-terminal regions of E1
and E2. The putative TMDs are underlined and charged
residues are indicated in bold. The sequence of the peptide
(E1-(350-370)) used for CD and NMR studies is indicated by an
open rectangle.
View larger version (47K):
[in a new window]
Fig. 2.
Effect of alanine insertions in E1 on the
assembly of the noncovalent E1·E2 complex. A, HepG2
cells were co-infected with vTF7-3 and the appropriate vaccinia virus
recombinant at a multiplicity of 5 plaque forming units/cell. At 4 h post-infection, cells were metabolically labeled for 10 min and
chased for 4 h. Cell lysates were immunoprecipitated with a
conformation-sensitive E2-specific mAb (H53) which recognizes the
noncovalent E1E2 heterodimer. A control immunoprecipitation with a
conformation-insensitive anti-E1 mAb (A4) was performed. Proteins were
separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide)
under reducing (A4) or nonreducing (H53) conditions. B, the
intensities of the bands corresponding to E1 and E2 proteins
precipitated by Mab H53 were measured by phosphorimaging for four
to eight independent experiments and the mean percentage of noncovalent
complex was calculated as follows for each mutant: (E1/E2 ratio from
mutated polyprotein)/(E1/E2 ratio from wild type polyprotein).
Positions of the alanine insertions in the TMD of E1 are
indicated.
View larger version (17K):
[in a new window]
Fig. 3.
Effect of alanine insertions in E2 on the
assembly of the noncovalent E1·E2 complex. See Fig. 2 for
legend.
View larger version (12K):
[in a new window]
Fig. 4.
HPLC analysis of the oligosaccharides bound
to CD4-E1-Ala358' (A) and E2-Ala727'
(B). HepG2 cells were co-infected with vTF7-3 and
the appropriate vaccinia virus recombinant at a multiplicity of 5 plaque forming units/cell. At 4 h post-infection, cells were
metabolically labeled for 30 min with [2-3H]mannose and
chased for 4 h. Cell lysates were immunoprecipitated with mAb OKT4
(anti-CD4) or H53 (anti-E2), and labeled glycans were removed by
peptide N-glycanase F treatment. M9, M8, and
M7 indicate the oligosaccharide species possessing two
GlcNAc residues at their reducing end and 9, 8, or 7 mannose residues,
respectively.
Folding of E1 Is Less Efficient When Heterodimerization Is Impaired
Since we have previously shown that E1 needs to be coexpressed
with E2 to fold properly (11, 16), we were interested to know whether
impairment in heterodimerization would lead to less efficient folding
of E1. E1 folding was analyzed for one of the most affected mutants
(Ala358') expressed in the context of an E1E2 polyprotein.
Disulfide bond formation in E1 was monitored by SDS-polyacylamide gel
electrophoresis under nonreducing conditions as described previously
(31). This method takes advantage of an increase in mobility as a
protein acquires a compact conformation stabilized by the formation of intramolecular disulfide bonds. An oxidized form of E1, which appeared
slowly, was clearly detected in the context of the wild type E1E2 (Fig.
5) as previously observed (31). In
contrast, the intensity of the oxidized form of E1-Ala358'
was lower, and a quantitative analysis showed a 40% reduction of
this form. It has to be noted that part of E1 separated under nonreducing conditions formed high molecular weight aggregates as a
function of time (data not shown), which explains the lower intensity
of the bands observed during the chase (Fig. 5). Together, these data
indicate that interaction of the TMDs of HCV envelope glycoproteins is
important for assisted folding of E1 by E2.
|
Structural Characterization of the E1 Region Involved in Heterodimerization
Previous reports have shown that alanine insertions disrupt interactions when introduced near the center of transmembrane helices (19, 20). Our data indicate that segments located close to the charged residue(s) present in the TMDs of E1 and E2 are directly involved in heterodimerization, suggesting that, despite their charge, these residues might be located near the center of the membrane spanning sequence (see "Discussion"). However, the presence of a second heterodimerization segment in the N-terminal region of the TMD of E1 is a novel finding which is not readily understood. To better understand the results observed for E1 mutants, the structure of a synthetic peptide identical to the sequence of the N-terminal part of the TMD of E1 (denoted E1-(350-370); see Fig. 1) was studied by CD and NMR. Indeed, CD and NMR spectroscopies can provide conformational information at the residue level on membrane proteins or isolated fragments, especially transmembrane segments incorporated into membrane environment or organic solvent (see Refs. 44-46 and references therein; for a review, see Ref. 47). For example, Shon et al. (48) have shown that the conformation of bacteriophage Pf1 coat protein with a single transmembrane segment is very similar in detergent micelles and phospholipid bilayer using solution and solid-state NMR spectroscopies, respectively. For larger membrane proteins Arseniev and colleagues (e.g. Refs. 49-51) have shown that the structure of isolated transmembrane spans of bacteriorhodopsin in the presence of organic solvents or detergent micelles is quite consistent with the structure of the corresponding regions in the whole protein determined by crystallography (52).
CD Analyses--
The secondary structure of E1-(350-370) was
readily examined by CD spectroscopy after solubilization in various
solvent systems which provide a membrane-like environment. Similar
approaches have been used in related studies (see above). Spectra shown
in Fig. 6 were obtained in three distinct
solvent systems in the presence of 10 mM sodium phosphate
(pH 7.4): 50% TFE, 1% lysophosphatidyl choline micelles, and 200 mM SDS micelles. In all cases, the CD spectra of
E1-(350-370) exhibited two minima around 208 and 222 nm, and a maximum
at 192 nm. This is typical of a peptide in -helix conformation.
Similar results were obtained when using dodecylmaltoside or
dodecylphosphocholine as membrane mimetic systems (data not shown).
Increasing the TFE concentration above 50% resulted in only minor
changes in the amplitude of the spectrum (data not shown), indicating
that the maximum -helical folding is reached in 50% TFE. This is
consistent with the suggestion of Jasanoff and Fersht (53) that, for a
peptide with high helical propensity, the helicity is complete by 50%
TFE. The maximal molar ellipticity per residue observed at 222 nm
corresponded to an -helical content of 48, 44, and 52% in TFE, SDS,
and LPC, respectively. These values are similar and clearly indicate
that E1-(350-370) is only partly folded into -helix. Moreover,
these results indicate that 50% TFE is an appropriate medium to mimic
the membrane-like environment of E1-(350-370) and validate its use to
analyze the conformation of this peptide by NMR (for an overview about
TFE, see Refs. 54 and 55).
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NMR Spectroscopy--
Deuterated micellar SDS and
dodecylphosphocholine are popular membrane mimetic solvents for
structure analysis of membrane peptides by liquid NMR (47).
Unfortunately, E1-(350-370) appeared to be poorly soluble in SDS or
dodecylphosphocholine in the millimolar range concentrations required
for NMR experiments. Consequently, we studied the three-dimensional
structure of E1-(350-370) dissolved in 50% TFE (v/v) which yielded
well resolved spectra as illustrated by the extract of NOESY (Fig.
7A). The spectra were assigned
using the classical method (38): the spin systems were identified with
DQF-COSY and TOCSY spectra. The sequential assignment was performed
with the help of the NOESY spectrum obtained at a mixing time of 150 ms. Despite the poor dispersion of the NH and H resonances (all NH
resonances are in a range of 0.9 ppm, and 17 of 21 H resonances are
in a range of 0.5 ppm), the sequential attribution of all spin systems
was completed (see Fig. 7B). No sign of any other stable
peptide conformer was observed in the NMR spectra. The 1H
chemical shifts are available under the BMRB accession number 4699.
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Secondary Structure--
Fig. 7B shows an overview of
the sequential and medium range NOE connectivities and the chemical
shift analysis for 1H. Despite the lack of data due to
numerous overlapping cross-peaks (indicated by asterisks on
Fig. 7B), the analysis of NOEs allows the distinction of
several structural regions. The main body of the peptide
(Ile359-Lys370 segment) displays most of the
characteristics of an -helix conformation, such as strong
dNN(i, i+1) and medium dN(i,
i+1) sequential connectivities, and weak dN(i,
i+2), medium dN(i, i+3), medium or
strong d(i, i+3), and weak
dN(i, i+4) medium range connectivities. The
C-terminal part of the peptide (Ala369 and
Lys370) remains more flexible with less medium range
connectivities, which is the sign of a fraying end often encountered in
peptide termini. The second structural unit (from Gly354 to
Gly358) is charaterized by stronger dN(i,
i+2) constraints and the lack of dN(i,
i+4) connectivities present in more flexible helical structures or that observed in a 3/10 helix. The N-terminal part of the
peptide (Gly350-Trp353) is devoid of medium
range NOEs and remains unstructured.
The 1H chemical shift difference
(
1H) is another NMR-related parameter used to
analyze the protein secondary structure, and it is negligibly affected
by addition of up to 50% TFE (56). Relative to a random conformation,
an increase of helicity results in an upfield shift of
1H resonances and a negative value of the corresponding
1H. In the central and C-terminal part of the
peptide (Gly354 to Trp370), the negative
1H is equal to or larger than 0.1 ppm. This is in
agreement with the criterion used to define an -helix conformation
(57) and confirms the helical conformation of this part of the peptide. The N-terminal part of the peptide
(Gly350-Trp353) fluctuates positively and
negatively, and no secondary structure could be unambiguously established.
Structure Calculation and Analysis--
In a first round, only
unambiguous NOE constraints were used for structure calculations. An
improvement of the constraints set was achieved through NOE
back-calculation that allowed the validation of all NOE-derived
distance constraints. Finally, a total of 337 interproton distance
constraints including 97 sequential and 105 medium range constraints
were used for the calculations (Table I).
No hydrogen-bond or angle constraints were introduced despite the clear
indications of an -helical folding. From the initial 50 structures
calculated with X-PLOR 3.1 (39), a final set of 24 low energy
structures was selected on the basis of no NOE violation greater than
0.5 Å. The pairwise comparison of these structures revealed only one
structural family. This is illustrated in Fig.
8A which shows a view of the
24 structures superimposed from the Ile359 to
Asn367. The overall energy of the 24 structures is largely
negative with values of less than 69 kcal mol
1 (288
kJ mol1). The stereochemical properties of the backbone
dihedral angles provided by the Ramachandran plots showed that all the
residues are located in the allowed regions (Table I). The parameters analyzing the deviation from the idealized covalent geometry are homogeneous. In summary, all the 24 structures fully satisfied the
experimental data and fit well with the finding of one family of
structures. The best representative structure of this family is
presented in Fig. 8B.
|
|
The -helix is well defined between residues Ile359 and
Asn367 as reflected by a low RMSD for the backbone heavy
atoms (C', C
, and N) of 0.31 Å and 1.03 Å when all the
heavy atoms are taken into account (Table I). Both the C- and
N-terminal parts of the peptide appear to be rather disordered.
However, some helical folding was observed on the C-terminal part of
the peptide (Trp368-Lys370 segment) in
calculated structures (Fig. 8A). This is in agreement with
the presence of some medium range NOE constraints in this region (see
Fig. 7). Concerning the N-terminal part, it should be noted that the
Gly354-Gly358 segment was mostly observed as
helical in each calculated structure (Fig. 8, B and
C), but failed to superimpose in all structures. This could
be related to the weak intensities of medium range -helix NOEs in
this region (see Fig. 7). This structural instability of the N terminus
of the peptide is very likely due to the presence of glycine residues
that are well known to be helix breakers. However, in the full-length
E1 glycoprotein context and/or E1E2 heterodimer context, it is very
likely that the stabilization of the conformation of this segment
arises from its interaction with other segments of these proteins. In
such a context, the presence of an helical folding of the
Gly350-Gly358 segment is possible. Fig.
8D shows a theoretical -helix projection of the
Ala349-Ile359 segment assuming that such a
conformation might occur in the full-length protein context. This
representation highlights that the three glycine residues 350, 354, and
358 are located on the same side of the putative -helix, suggesting
a role for these glycines in E1E2 heterodimerization.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The TMDs of HCV envelope proteins are extreme examples of multifunctionality of membrane spanning sequences. Indeed, besides their role as membrane anchor, they possess a signal sequence function in their C-terminal half, are responsible for ER localization of E1 and E2, and play a role in the assembly of these proteins. In this work, we used alanine scanning insertion mutagenesis to study the oligomerization function of these domains. We demonstrated that the TMDs of E1 and E2 play a direct role in heterodimerization and that E1E2 assembly can be disrupted without affecting the other functions. Moreover, whereas one segment was found to be critical for heterodimerization in the TMD of E2, two structurally different segments were identified for E1. In addition, we determined the three-dimensional structure of E1-(350-370) peptide in the presence of membrane mimetic solvents by NMR to provide a structural framework for a better analysis of the mechanism of E1E2 heterodimerization.
The TMDs of HCV envelope glycoproteins play a direct role in
heterodimerization. Deletion or replacement of the C-terminal hydrophobic sequence of E2, or mutation of the charged residues in the
TMDs of E1 or E2 have been shown to dramatically reduce the formation
of E1·E2 complexes (3, 11, 15, 18). Although these data indicate that
the TMDs are involved in the assembly of HCV envelope glycoproteins,
the effect of the mutations of charged residues might be indirect.
Indeed, these mutations lead to secretion or transport of the mutated
protein out of the ER compartment, and the lack of complex formation
could be due to the impossibility for E1 and E2 to find each other at
the time of assembly. However, E1 and E2 interact before completion of their folding (9), and this process is supposed to occur in a specific
compartment, the ER (58). The impairment in heterodimerization following alanine scanning insertion mutagenesis clearly shows that the
TMDs of E1 and E2 are directly involved in assembly. Indeed, this
technique has been shown to be very useful to identify critical
segments of transmembrane -helices involved in helix-helix interactions (19, 20). In addition, data of alanine scanning insertion
mutagenesis (20, 59) fit very well with a recent model predicting the
stability of transmembrane helix-helix interactions in mutants of
glycophorin A (60). Interestingly, with the exception of the impairment
in assisted folding of E1 by E2, no other functions of the TMDs of HCV
envelope glycoproteins seems to be altered. This is clearly different
from replacement mutagenesis of the charged residues which leads to an
alteration of all the functions played by these TMDs (3).
The TMDs of E1 and E2 are probably not the sole determinants for heterodimerization. N-terminal sequences in E2 and also in E1 have been suggested to be important for HCV envelope glycoprotein assembly (61), but deletion mutant analysis has failed to identify any single region which is required for noncovalent interaction (62). Interestingly, assisted folding of the ectodomain of E1 by E2 suggests that regions other than the TMDs might enter into contact. For many viral envelope proteins, the ectodomains have been shown to be involved in oligomerization (63), a feature which is important to regulate the fusogenic function of these proteins (64). Why should there be different regions involved in heterodimerization of HCV envelope glycoproteins? Assembly and folding of HCV envelope glycoproteins seem to be interconnected events. It is likely that an early contact between these proteins, probably initiated by their TMDs, is necessary to bring their ectodomains into contact, which seems to be necessary for the formation of a native complex. This could explain why an alanine insertion mutation which is disruptive for assembly is also disruptive for the assisted folding of E1 by E2.
The data of alanine scanning insertion mutagenesis in the TMD of E2
suggest that this domain forms a single transmembrane segment with the
charged residues located in the middle of the membrane spanning
sequence. This fits well with the prediction of a single transmembrane
helical segment from residue 718 to 742. Indeed, a minimum of 16 leucines are required to form a transmembrane -helix (65), but
stretches of 20-25 hydrophobic amino acids are generally observed in
integral membrane proteins. Although the presence of charged residues
within the center of this transmembrane helix seems to be surprising
a priori, it is very unlikely that segment 718-742 adopts a
stable conformation allowing two transmembrane passages. In such
eventuality, each of the two small hydrophobic stretches (718-727 and
731-742) would have to adopt an extended structure. Such an extended
structure is very unfavorable in a membrane environment because of the
absence of stabilization by hydrogen bonds, while the hydrogen-bond
network of an -helix offers the most stable solution from a
thermodynamic standpoint. The formation of transmembrane -sheets as
observed in the porin family of membrane proteins also yields a stable
hydrogen-bond network compatible with the membranous environment.
However, examination of the nature and position of residues in the TMD
of E2 does not provide any sign that such a stable conformation might
take place. Concerning the potential problem of the 2 charged residues
within the membrane (Asp and Arg), it is possible that they could form ion pairs as already described in the cases of several membrane proteins (66). Taken together, these data clearly support the hypothesis that the TMD of E2 is a single transmembrane helix centered
near amino acid 730.
Concerning the TMD of E1, the data of alanine scanning insertion
mutagenesis allowed us to identify two distinct segments involved in
heterodimerization with the TMD of E2. First, similarly to what was
observed for the mutations in E2, the disruptive effect of
Ala369' inserted very close to Lys370 suggests
that this charged residue might also have a central position in the
membrane. Second, the N-terminal region of the TMD of E1 appears to be
very sensitive to alanine insertions. This is likely due to the
presence of GXXXG motifs which have been well documented to
ensure specific homodimerization of transmembrane segments in membrane
proteins (see below). These features, together with the thermodynamic
reasons exposed above for the TMD of E2, strongly suggest that E1 is
anchored in the membrane by a single transmembrane -helix. This is
also supported by the fact that in the E1-(350-370) model (see Fig.
8), the Ile359-Asn367 segment was found to form
a stable -helix and the Gly354-Gly358
segment has a clear tendency to adopt an helical fold (see Fig. 8). In
addition, taking into account that aromatic residues and positively
charged residues are often encountered at the membrane interface, it
might be predicted that His352 and Trp353
residues form the N terminus of the TMD of E1. In summary, we thus
assume that the 25-amino acid long segment
Gly354-Phe379 forms a single transmembrane
helix and is the counterpart of the E2 transmembrane helix. It should
be underlined that a single membrane spanning topology for the TMDs of
E1 and E2 would also fit very well with the current model of ER
retention by membrane determinants. A usual feature of such signals is
the presence of one or several hydrophilic residues within the
hydrophobic TMD (67-70), and their effect is strongest when they are
localized toward the middle of the membrane (67).
To sequentially ensure some of their functions, the TMDs of E1 and E2 might adopt different topologies. Besides their role in heterodimerization, these TMDs also have a signal sequence function in their C-terminal half (3). Since the ectodomains of E1 and E2 are translocated into the lumen of the ER, the N terminus of their TMD should also be oriented toward the luminal side of the ER. The signal sequence function present in the second half of these TMDs suggests that the C terminus of these domains should also be oriented, at least transiently, toward the luminal side of the ER. These domains are therefore likely to form a hairpin structure with the charged residues close to the cytosolic face of the membrane. It is reasonable to think that this hairpin structure might be transient in the environment of the translocon before signal sequence cleavage has occurred. A reorientation of the second stretch of hydrophobic residues leading to a single transmembrane segment would occur immediately after signal sequence cleavage at its C terminus and before membrane integration and heterodimerization.
Assuming that the TMDs of E1 and E2 are both composed of a single
transmembrane -helix, it is reasonable to postulate that these 2 helices interact together to ensure E1E2 heterodimer formation. However, the putative existence of a single transmembrane -helix between Gly354 and Phe379 suggests that part of
the Gly350-Gly354 segment might lie outside of
the membrane while it is sensitive to alanine insertion. From a
structural point of view, this segment appears to be unfolded (see Fig.
8), but this is likely due to the absence of conformational
stabilization by tertiary interactions in the isolated E1-(350-370)
peptide context used here. In the E1 and/or E1E2 context, it is thus
possible that the Gly350-Gly354 segment folds
into an -helix and that the whole E1-(350-379) segment forms a
single long -helix upon E1E2 heterodimerization. Interestingly, the
simulation of such conformation for the
Gly350-Gly358 segment clearly shows that the
three well conserved glycine residues (Gly350,
Gly354, and Gly358) lie on the same side of the
putative helix (see helix projection, Fig. 8D) and form 2 consecutive putative GXXXG motifs
(Gly350-Gly354 and
Gly354-Gly358). The presence of such a glycine
motif has been reported to be essential to ensure the specific helix to
helix interaction for several transmembrane proteins such as
glycophorin A (71) and phage M13 coat protein (72). Moreover, the
importance of the GXXXG motif has been recently highlighted
by two articles of Engelman and colleagues (73, 74): (i) in one
experimental study, they have selected TMDs exhibiting high affinity
homo-oligomerization from a randomized sequence library based on the
right-handed dimerization motif of glycophorin A and this
GXXXG motif was the most frequent isolated using the TOXCAT
system which measures transmembrane helix-helix association in the
Escherichia coli inner membrane (73); (ii) in a theoretical
study, they have analyzed frequently occurring combinations of residues
in a data base of putative TMDs and the main theme observed is patterns
of small residues (Gly, Ala, and Ser) at i and
i+4 found in association with large aliphatic residues (Ile,
Val, and Leu) at neighboring positions (i.e.
i+/1 and i+/2) (74). A very suggestive
correlation can be made with our studies: coinciding with the
most dramatic effects of Ala insertion, the TMD of E1 displays in its
N-terminal part two successive GXXXG motifs, and the second
one is GVXXGI. Consequently, it is tempting to speculate
that the 2 glycine motifs of E1 play a major role for E1E2-specific
heterodimerization and are located at the interface of E1 and E2 dimer.
In conclusion, the structural and functional analyses reported here
allowed us to show for the first time that oligomerization of viral
envelope proteins can be controlled by transmembrane sequences and to
propose a molecular framework for the understanding of the mechanism of
E1E2 heterodimerization. Besides the comprehension of the assembly of
HCV envelope glycoproteins, the multifunctionality of the TMDs of E1
and E2 provides a very interesting model to study the
structure/function relationship of transmembrane sequences. The
functions currently identified for these proteins are taking place
during the early events of E1E2 synthesis and assembly. Such a model
will also be very helpful to understand the complexity of the dynamic
relationship between neosynthesized proteins and the translocation machinery.
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ACKNOWLEDGEMENTS |
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We thank Françoise Jacob-Dubuisson for critical reading of the manuscript, André Pillez and Sophana Ung for excellent technical assistance, Pierre Falson, Czeslaw Wychowski, Christophe Combet, and Gilbert Deléage for stimulating discussions, and Graham Blomberg for peptide synthesis.
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FOOTNOTES |
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* This work was supported by the CNRS, Institut Pasteur de Lille, European Regional Development Fund (ERDF), a PRFMMIP grant from the French Ministry of Research, European Union Grant QLK2-1999-00356, and Association pour la Recherche sur le Cancer (ARC) Grant 9736. Support was also provided by a fellowship from ARC and the Agence Nationale de la Recherche sur le Sida (ANRS) (to A. O. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates for the NMR structure of the E1-(350-370) fragment and the NMR restraints are available in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession number 1EMZ (RCSB010735). The proton chemical shifts of all residues have been deposited in the BioMagResBank (BMRB) under the accession number 4699.
Contributed equally to the results of this study.
§§ To whom correspondence should be addressed: Equipe Hépatite C, CNRS-UMR8526, Institut de Biologie de Lille & Institut Pasteur de Lille, 1 rue Calmette, BP447, 59021 Lille cedex, France. Tel.: 33-3-20-87-11-60; Fax: 33-3-20-87-11-11; E-mail: jean.dubuisson@ibl.fr.
Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M003003200
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ABBREVIATIONS |
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The abbreviations used are: TMD, transmembrane domain; HCV, hepatitis C virus; mAb, monoclonal antibody; DQF-COSY, double quantum-filtered correlation spectroscopy; LPC, lysophosphatidylcholine; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root mean squared deviation; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy; HPLC, high-pressure liquid chromatography; ER, endoplasmic reticulum.
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REFERENCES |
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