 |
INTRODUCTION |
Heat-labile enterotoxin
(Etx)1 is a hexameric protein
produced by certain diarrheagenic strains of Escherichia
coli and is closely related to cholera toxin in both structure and
mode of action (for a review, see Ref. 1). The holotoxin has an
AB5 structure, consisting of one A subunit (EtxA, 240 amino
acids, 28 kDa) which catalyzes the ADP-ribosylation and activation of the G-protein Gs
, and five identical B monomers (EtxB,
103 amino acids, 11.6 kDa each) which are assembled in a pentameric
structure and bind to the eukaryotic cell-surface receptor,
monosialoganglioside GM1 (2).
The pentameric B subunit moiety of Etx can be readily purified in large
quantities, and its stability, non-toxic properties, and ability to
bind to ubiquitous GM1 receptors make it a potential delivery vehicle
for heterologous peptides (3-5). Recently, we constructed a
recombinant fusion protein (EtxB-R2) comprising the enterotoxin B
subunit derived from E. coli H74-114 of human origin linked
to the COOH-terminal 9 amino acids of the small subunit (R2) of
ribonucleotide reductase of herpes simplex virus type 1 (HSV-1). The
attached nonapeptide inhibits ribonucleotide reductase activity
in vitro by disrupting the functional association between
the enzyme subunits (6-8). We showed that the EtxB-R2 chimera could
specifically inhibit viral replication in HSV-1-infected Vero cells,
implying the EtxB-mediated delivery of the nonapeptide to the cytosol
and inhibition of ribonucleotide reductase activity (5).
More recently, we also reported the construction of another recombinant
toxin chimera (EtxB-Pol) derived from fusion of the etxB
gene with a sequence encoding the 27 COOH-terminal amino acids of the
catalytic subunit (POL) of HSV-1 DNA polymerase (9, 10). The
carboxyl-terminal region of POL is responsible for its association with
UL42, a smaller subunit which is not necessary for basal activity but
increases both the rate of incorporation of deoxyribonucleotide
triphosphates and the processivity of the enzyme (11, 12). The EtxB-Pol
chimera retained the functional properties of both components, being
able both to bind to the GM1 cell receptor like the wild type EtxB, and
associate to UL42 as HSV-1 POL (10). On the basis of our data, we
proposed the use of EtxB as a protein-based carrier system for the
delivery of heterologous peptides in the intracellular compartment
(5).
These emerging biotechnological applications of the EtxB-based chimeric
toxins have highlighted the need for understanding of their
three-dimensional structure. So far, the crystal structures of the
AB5 holotoxin from an E. coli strain of porcine
origin (13-15), of its complexes with lactose (16), galactose (17), with a tumor marker disaccharide (18) and of two mutants (19, 20) are
already known, along with those of the structurally related cholera
toxin (21). Moreover, the crystal structure of complexes of the wild
type B subunit with
D-galactopyranosyl-
-D-thio-galactopyranoside and meta-nitrophenyl-D-galactopyranoside has
been solved (22). The structures of the cholera toxin B subunit (23),
and the structurally related E. coli verotoxin-1 (24) have
also been published.
In this paper, the crystal structures of the EtxB-R2 and EtxB-Pol
chimeric proteins at 3-Å and 3.3-Å resolution, respectively, are
described. Moreover, structural characteristics are correlated with
intracellular processing of chimeras and with release of biologically
active peptides.
 |
MATERIALS AND METHODS |
Proteins--
The hybrid EtxB-R2 protein, encoded by plasmid
pAM320, was obtained by the genetic fusion of the nine COOH-terminal
amino acids (YAGAVVNDL in single letter code) of the small subunit of HSV-1 ribonucleotide reductase to the etxB gene, as
described elsewhere (5).
The EtxB-Pol fusion protein, encoded by plasmid pAL3, was created
by amplifying the region of pE30 (25) corresponding to the 27 COOH-terminal amino acids of HSV-1 POL using the polymerase chain
reaction and then inserting this fragment after the etxB gene, as reported (10).
Both EtxB-R2 and EtxB-Pol were expressed in marine non-toxinogenic
Vibrio sp. 60 (strain MTV606, obtained from Dr. A. Ichige, University of Tokyo), and purified as reported previously (10, 26),
with minor modifications.
Mass Determination--
50 µg of purified EtxB-R2 protein was
desalted with a Supelcosil LC318 analytical C18 column, using a
gradient of 20-80% CH3CN (0.05% trifluoroacetic acid) in
20 min. Mass spectra were measured with an electrospray ionization
spectrometer (Perkin-Elmer Sciex API-1), on the sample directly eluted
from the reverse phase column. Mass scans were accumulated in positive
mode, with ionization and orifice voltages of 5000 V and 90 V,
respectively, and a resolution of 0.1 mass units, in the 1200-2400
mass range. The mass peak reconstruct was obtained via the Fenn method
(PE-Sciex, API-1 user manual).
Immunofluorescence and Microscopy--
Cells for
immunofluorescence were plated into 24-wells trays at a density of
6 × 104 cells per well, containing one coverslip per
well and grown for 24 h. After treatment with toxin, cells were
fixed with 3% paraformaldehyde in phosphate-buffered saline for 20 min
at room temperature, treated with 0.27% NH4Cl, 0.38%
glycine for 10 min, and permeabilized with 0.2% saponine, 0.5% bovine
serum albumin in phosphate-buffered saline for 30 min. Primary
antibodies were diluted in the permeabilization medium and applied to
cells for 1 h. The monoclonal antibody 118-8 anti-EtxB was kindly
provided by Drs. E. Lundgren and H. Persson (University of Umea,
Sweden), whereas the polyclonal antibody anti-EtxB was obtained from
Dr. M. Pizza (27). The polyclonal antiserum 113 was raised against the
COOH-terminal 15 residues of HSV-1 POL (28). mAb 8746 (supplied by
Hilkka Lankinen, Institute of Virology, Glasgow) was raised against the
YAGAVVNDL peptide. After several washes, Texas Red- or
fluoresceine-conjugated secondary antibodies (from Jackson
Immunoresearch Laboratories and Calbiochem, respectively) were added,
incubated for a further 30 min in the same medium, and then washed.
Samples were mounted in 90% glycerol, 0.2% N-propylgallate
in phosphate-buffered saline, and observed by fluorescence microscopy.
Fluorescent optical sections (1 µm thick) of cells were obtained with
a Zeiss Axiovert TV-100 fluorescence microscope, equipped with a CD
camera, using the Metamorph deconvolution software.
Crystallization--
Crystals of EtxB-R2 and EtxB-Pol were
obtained with the hanging drop method. 2 µl of protein solution,
approximately 7 mg/ml, were mixed with the same volume of the
precipitant solution containing 0.8 M lithium sulfate and
2% PEG 8000. The drops were equilibrated against 0.5 ml of the same
precipitant solution. Needle shaped crystals of about 0.6 × 0.2 × 0.1 mm3 grew in 2-3 weeks at 4 °C, but were
unstable and tended to dissolve, possibly owing to the large solvent
content. Both structures crystallize in the tetragonal space group
P41212, with similar parameters of the unit
cell of a = b = 127.23, c = 174.19 Å for EtxB-R2, and a = b = 127.10, c = 176.10 Å for EtxB-Pol.
Structure solution has demonstrated the presence of one pentameric
molecule per asymmetric unit, corresponding to Vm values (29)
of 5.5 and 4.8 and to a solvent content of about 77 and 74% for
EtxB-R2 and EtxB-Pol, respectively.
Structure Solution and Refinement--
Despite their reasonable
size, crystals did not diffract under a conventional x-ray source. Data
were consequently measured at the ELETTRA synchrotron in Trieste
(Italy), using an imaging plate detector system (MAR Research), with a
diameter of 300 mm. Immediately before data collection the crystal was
picked up with a loop, dipped for a very short time in a cryoprotectant
solution, obtained by mixing 67% of the precipitant solution with 33%
glycerol (v/v), and frozen in a nitrogen vapor stream at 100 K. The
temperature was controlled by an Oxford Cryosystems Cryostream. Data
were processed with MOSFLM (30). Statistics on data collection and processing are reported in Table I. The
structure of EtxB-R2 was solved with the molecular replacement
technique and the AMoRe software (31) from the CCP4 suite (32), using
as a template the coordinates of the B subunit of porcine E. coli heat-labile enterotoxin extracted from the AB5
complex (1LTS, Ref. 14). The first five maxima of the rotation
function, all related by a 5-fold symmetry, presented correlation
coefficients between 13.7 and 11.6. The difference to the subsequent
maximum of 11.3 was small. The highest peak of the translation function
corresponded to a correlation factor of 62 and to a crystallographic
R factor of 0.37. Despite an accurate search, it was
impossible to fit a second pentameric molecule in the crystal cell.
Visual inspection of the only structure solution obtained showed a
reasonable three-dimensional packing, despite large holes present in
the crystal lattice, and no clashes with symmetry-related molecules
(Fig. 1). The structure was subjected to
a rigid-body refinement procedure that brought the crystallographic
R factor to 0.34. From this point onwards, visual
inspections of the electron density maps and manual rebuilding using
the TOM program (33) were alternated with automatic minimizations, for
a total of 43 macrocycles. Differences in the electron density could be
seen already in the first few cycles, in correspondence to residues 13 (His in human and Arg in porcine strain E. coli EtxB) and 46 (Ala instead of Glu). The other 2 amino acids that differ in the human
and porcine strain sequences (Ser instead of Thr in position 4 and Glu
instead of Lys in position 102), could not be unambiguously
distinguished at this resolution. Moreover, some density was also
visible close to the COOH-terminal end of each monomeric chain,
corresponding to the peptide extension, but was difficult to interpret
in terms of the known amino acid sequence. In the course of the
refinement this density became clearer and some, but not all, residues
of the extension could be fitted (see "Results and Discussion").
116 solvent molecules were positioned as water, along with 6 sulfate
ions. The final crystallographic R factor was 0.18 (Rfree 0.22).

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 1.
Stereo drawing of the packing of the EtxB-R2
fusion protein in the crystal cell seen along the crystallographic
c axis. Only C atoms are drawn for each
molecule. A similar packing is present in the EtxB-Pol crystals.
|
|
During the positional and thermal factor refinement, performed with the
X-PLOR program (34, 35), non-crystallographic symmetry was imposed to
main chain and side chain atoms, with different weights. The atoms
corresponding to the extension were excluded from this symmetry and
allowed to refine independently. They were assigned an occupancy factor
of 0.75.
The molecular model determined for EtxB-R2, deprived of the amino acids
corresponding to the peptide extension and of the solvent molecules,
was used as the starting point for the refinement of the EtxB-Pol
structure. 32 cycles of minimization performed with the X-PLOR program,
alternated with inspections of the electron density maps and manual
rebuilding, brought the R factor to 0.21 (Rfree = 0.25). An examination of the electron
density maps, however, allowed positioning of only a few ordered amino
acids. Statistics on the final models are reported in Table
II.
 |
RESULTS AND DISCUSSION |
EtxB-R2--
The final molecular model consists of 4230 protein
atoms, 6 sulfate ions, and 116 water molecules: a control of the
stereochemistry performed with the PROCHECK program (36)
indicates that only 1 residue (0.2%) falls in the
"disallowed" regions, 99.0% of the residues fall in "favored"
or "allowed" regions, and 0.8% in the "generously allowed"
regions. All the other indicators are also consistent with or better
than those expected for a structure at 3-Å resolution. The only areas
that are not well ordered are loop residues 54-61. A special
case is represented by the peptide extension, which is discussed in
detail below.
The five polypeptide chains that form the pentamer, numbered in our
model from D to H (Fig. 2), assume quite
a similar conformation and are related by a 5-fold non-crystallographic
symmetry axis. A restraint on the non-crystallographic symmetry
was imposed during the refinement, with the exception of the loop
comprising residues 54-61, as differences were evident in the electron
density map among different monomeric chains (Fig.
3), and for side chains involved in
intermolecular contacts.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 2.
Schematic drawing of the overall structure of
the EtxB-R2 pentamer, prepared with the MOLSCRIPT program (37).
The portion of peptide extensions visible in the electron density map
are in black. Arrows indicate the loop 54-61.
The amino acid sequences of COOH terminus of the two fusion proteins,
from residue 102, are (peptide extensions in bold letters):
EtxB-R2, ...
Glu-Lys-Leu-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu;
EtxB-Pol, ...
Glu-Lys-Leu-Ala-Gly-Phe-Gly-Ala-Val-Gly-Ala-Gly-Ala-Thr-Ala-Glu-Glu-Thr-Arg-Arg-Met-Leu-His-Arg-Ala-Phe-Asp-Thr-Leu-Ala.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Root mean square deviations of
C atoms of one monomer of the B subunit
pentamer (residues 1-105) with respect to the other four polypeptide
chains. Significant differences are present only around residues
54-61 and from 102 to 105.
|
|
The overall conformation of the EtxB-R2 pentamer, schematically shown
in Fig. 2, is very similar to the wild type EtxB pentamer of the
porcine strain E. coli enterotoxin (14); five subunits, each
consisting of two antiparallel
-sheets and two
-helices, are
arranged around a central 5-fold axis. A comparison of these two
pentamers gives an average root mean square deviation between our
model, from residues 1 to 103, and the corresponding residues of the
wild type of 0.32, 0.40, 0.44, 0.62, and 0.46 Å for monomers D to H,
respectively. The only significant variation between EtxB from E. coli strains of porcine and human origin is detectable around
residues 54 to 61, corresponding to a solvent exposed loop. As some
differences have been reported for receptor-binding specificity and
antigenic determinants (38, 39), it could be proposed that these could
in part depend from the exposed loop.
The amino acid sequence of our chimeric protein corresponds to the
sequence of native EtxB up to position 102. Asn103 of the
original sequence is substituted by Lys in the chimera, a Leu has been
added at position 104 by the cloning procedure, and amino acids 105 to
113 correspond to the attached R2 nonapeptide (see legend to Fig. 2).
This extension presents common features in all monomeric chains only
for residues 103-105 (Fig. 4), as the
-strand that spans residues 95-100 in the native protein extends
roughly to residue 105 in the chimera. The side chains of
Lys103 and Tyr105 point toward the interior of
the molecule, and Leu104 toward the solvent. The
substitution of a polar amino acid (Asn) in position 103 with a
positively charged Lys introduces a peculiar feature in the molecule: a
peak of electron density, strictly obeying the molecular 5-fold
symmetry, is present close to N
of Lys103 and to N of
Lys81. Owing to its size and to the presence of the two
positive charges, it has been interpreted as a sulfate ion, due to the
presence of lithium sulfate in the crystal mother liquid. We can
suppose that in solution these two positive charges will be neutralized by some other negative ion. From residue 105 onwards, the situation differs in all chains. In monomers E, G, and H the remaining portion of
the polypeptide could not be positioned in our model. In monomer E a
large and confused density can actually be seen close to the COOH
terminus and to the symmetry related molecule, but there is a gap
between residue 105 and this density. For this reason, we preferred not
to interpret this portion. On the contrary, in monomer D amino acids
104-108 could be positioned in the electron density map: residues 105 and 106 form a sort of tight turn, so that residues 107 and 108 continue in the reverse direction with respect to the preceding
-strand. In monomer F, the polypeptide chain is ordered up to
residue 109, but in this case the final part does not make a reverse
turn and can better be described as an extended chain. The reason of
the different behavior of the COOH-terminal residues of the latter two
chains can be found in the fact that the final part of the extension in
monomer F makes close contacts with a symmetry-related pentamer.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4.
Stereo drawing of the C
chain trace of the EtxB-R2 subunit, with all the atoms
superimposed for each extension: amino acids from 103 to 108 in chain
D, from 103 to 109 in chain F, from 103 to 105 in chains E, G, and
H. The five sulfate ions close to Lys103 are also
shown.
|
|
In conclusion the entire peptide extension cannot be clearly
distinguished in any of the five monomers of the chimera, and the most
likely explanation is that only a few residues in the extensions are
ordered in the pentamer. The eventuality that portions of the
COOH-terminal chains were absent from the crystal structure due to
proteolytic degradation during the purification process was excluded on
the basis of mass spectrometric results. These demonstrated the
presence of a single species with mass 12729.5 Da, corresponding to the
theoretical mass (12730.6 Da) of an EtxB-R2 monomeric chain (Fig.
5). Moreover, it would have been
difficult to reconcile proteolytic cleavage at different positions with the crystal symmetry.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 5.
Electrospray mass spectrum (A) of
the EtxB-R2 monomer. The different peaks represent the indicated
charge states of the molecule. The measured molecular weight is shown
in the reconstructed spectrum (B).
|
|
An important characteristic resulting from the three-dimensional
structure of the chimeric protein is that the presence of the peptide
extension has no effect on the overall three-dimensional structure of
the B subunit pentamer. In fact, the structure of the chimera resembles
that of the B subunit pentamer bound to the A subunit in the native
protein. Moreover, the extension is fully confined to the surface of
the pentamer opposite to the face that interacts with the membrane
(14). Thus, peptide chains that extend from the COOH-terminal end of
the protein should not interfere with the ability of the B subunit
pentamer to interact with membrane gangliosides, as previously
suggested by enzyme-linked immunosorbent assay experiments with soluble
GM1 (40).
EtxB-Pol--
The data for the EtxB-Pol structure were obtained
only at 3.3-Å resolution and the crystal gave a weaker diffraction
pattern than that of EtxB-R2, resulting in a much smaller data set.
Therefore only the main features will be described. No sulfate ions
could be detected and solvent molecules were not included in the model, which consists of 4191 non-hydrogen atoms. No more than 3-4 residues for each chain, corresponding to amino acids 104-107, could be safely
fitted in the electron density maps. Some other electron density was
visible, but not connected to the main chain and not clearly
interpretable. In this case also, the B subunit pentamer maintains the
doughnut-shaped structure of wild type EtxB, even if the attached POL
peptide represents more than one-fourth the length of each B monomer.
The 36 COOH-terminal amino acids of POL have been predicted to
adopt a structure consisting of two
-helical regions interrupted by
a non-helical segment (41). We cannot exclude, owing to the quite low
resolution of our electron density map, that a portion of the
POL-derived peptide extension in our chimera could assume a definite
conformation, despite being orientationally disordered with respect to
the EtxB core.
Intracellular Processing of EtxB-R2 and EtxB-Pol--
To study the
intracellular fate of the two chimeras, Vero cells were treated for
different times with 10 µM EtxB-R2 or EtxB-Pol and
then stained either with anti-EtxB polyclonal and anti-R2 monoclonal antibodies, for EtxB-R2, or with anti-EtxB monoclonal and
anti-Pol peptide antiserum, for EtxB-Pol. Analysis by deconvolution fluorescence microscopy showed that both EtxB-R2 and EtxB-Pol bound to
cell surface receptors and were internalized in a manner resembling
that of wild-type EtxB. These results confirm that addition of such
peptides to the EtxB COOH terminus does not interfere with the receptor
binding properties of either EtxB-based fusion protein in a cell
system, and is consistent with our observations on the
three-dimensional structure.
After 10 min incubation the two domains of both fusion proteins fully
co-localized in punctated intracellular compartments which appeared
either scattered throughout the cytoplasm or in the perinuclear area
(Fig. 6, A, panels a, b, and
c and B, panels g, h, and i),
indicating that after internalization both EtxB-R2 and EtxB-Pol fusion
proteins are initially intact. After 30 min the toxin and peptide
portions of EtxB-Pol still co-localized (data not shown), whereas the
two moieties of EtxB-R2 were beginning to dissociate: in addition to
EtxB+/R2+ compartments in the perinuclear region, numerous EtxB+/R2
vesicles were visible in the surrounding cytoplasm (Fig. 6A,
panels d, e, and f). A similar pattern for EtxB-Pol,
with dissociation of its two components, was detectable after 1 h
of treatment (Fig. 6B, panels l, m, and n).

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 6.
Intracellular processing of EtxB-based fusion
proteins. A, Vero cells were treated with EtxB-R2 for
10 min (panels a, b, and c) or 30 min
(panels d, e, and f) and probed with a polyclonal
antibody specific for EtxB (EtxB, left panels)
and with the monoclonal antibody 8746, specific for R2 peptide
(R2, central panels) and secondary anti-rabbit
fluoresceine-conjugated or anti-mouse Texas Red-conjugated antibodies,
respectively. Images were collected using a × 100 objective with
a deconvolution fluorescence microscope and superimposed
(EtxB/R2, right panels). A yellow color indicates
colocalization of the two domains. Arrows and
arrowheads point at representative EtxB+/R2+ and EtxB+/R2
compartments, respectively. B, Vero cells were treated with
EtxB-Pol for 10 min (panels g, h, and i) or
1 h (panels l, m, and n) and probed with
monoclonal antibody 118-8, specific for EtxB (EtxB, left
panels) and with polyclonal antiserum 113, specific for POL
peptide (Pol, central panels) and secondary anti-mouse
fluoresceine-conjugated or anti-rabbit Texas Red-conjugated antibodies,
respectively. Images were analyzed as described in A.
Arrows and arrowheads point at representative
EtxB+/Pol+ and EtxB+/Pol compartments, respectively.
|
|
Taken together, these results indicate that the EtxB can mediate the
intracellular delivery of both R2 and Pol peptides, initially bound to
EtxB and later as free molecules which are proteolytically cleaved from
the toxin moiety. The decrease of the R2 signal at later times suggests
that the R2 peptide may translocate in the cytosol causing a signal
dilution. A complete degradation of the nonapeptide by cellular
proteases should be excluded, since we previously showed that the
addition of the EtxB-R2 fusion protein to virally infected Vero cells
resulted in the specific inhibition of HSV-1 replication and in a
reduction in dTTP levels (5), indicative of the inhibition of viral
ribonucleotide reductase which is located in the cytosol. Moreover,
preliminary studies demonstrated that EtxB-Pol is also active in
inhibiting HSV-1 replication in Vero
cells.2
Conclusions--
The two structures presented in this article
share a common conformational pattern: a highly rigid molecular core
and a very flexible peptide extension. The latter is effectively
floating in the solvent and is thus easily cut by a protease. This is
most likely what occurs in the cell, as suggested by the fluorescence microscopy experiments. From our model, a putative site of proteolytic cleavage is located between residues 105 and 106. A break at this position would produce peptides of 8 and 26 residues, respectively, for
EtxB-R2 and EtxB-Pol, with the correct sequence for interacting with
the HSV-1 ribonucleotide reductase in the first case, and with DNA
polymerase in the second. In fact, our data on the inhibitory activity
of both EtxB-R2 (5) and EtxB-Pol2 suggest that after
intracellular proteolytic cleavage, a sufficient number of molecules of
either peptide survives long enough to affect the viral target. The
absence of an ordered structure for the attached peptides may thus
represent an advantage, as it allows the extension to be cleaved by a
cellular endoprotease, freeing it to interact with the viral target
protein. In conclusion, the structural observations presented in this
article further support the idea that EtxB could be used as a
protein-based carrier system (5) for the intracellular delivery of
heterologous peptides.