Diphtheria toxin is secreted by Corynebacterium diphtheriae as a single polypeptide of 535 residues (M
58,348)(1) . The polypeptide chain is easily cleaved by
proteases into the 193-residue A chain (M
21,167),
which is located at the amino-terminal end of the protein, and the
342-residue B chain (M
37,199) at the
carboxyl-terminal end. The A chain's function is to catalyze the
transfer of the ADP moiety of NAD
to elongation
factor-2. This inhibits protein synthesis and causes cell
death(2) . The B chain has at least two functions: to aid in
translocation of the toxin across a lipid bilayer and in binding to the
cellular receptor(2) .
The crystal structure of diphtheria
toxin at neutral pH shows that toxin comprises three domains: a
catalytic (C),
a transmembrane (T), and a receptor-binding
(R) domain(3) . The catalytic domain is identical to the A
chain and largely comprises
-strands and short helices. The T
domain is the amino-terminal half of the B chain and largely comprises
long helices, several of which are hydrophobic. The R domain is the
carboxyl-terminal half of the B chain and comprises largely
-strands.
The toxin first binds to the cell and then enters by
receptor-mediated endocytosis(4) . Once endocytosis has
transported the toxin to endosomes, the acidic lumen of the endosomal
vesicle triggers a conformational change in the toxin causing it to
insert into the membrane bilayer(4) . After insertion into the
bilayer, the A chain translocates across the bilayer and is released
into the cytoplasm where it catalyzes ADP-ribosylation.
In previous
studies we have found that diphtheria toxin undergoes a conformation
change to a hydrophobic, molten globule-like structure at low
pH(5, 6) . When toxin is exposed to low pH at 23
°C in the presence of lipid vesicles, only the B chain undergoes
unfolding. However, when toxin is exposed to low pH at higher
temperatures, both the A and B chain unfold (7) .)
We chose anti-toxin antibodies to further examine diphtheria
toxin structure in order to identify the regions of the toxin that
undergo conformational changes at low pH. To do this, these antibodies
were examined for their ability to bind to the different conformations
of diphtheria toxin. The results suggest that all three domains undergo
some type of structural change at low pH. Combined with the results of
the following paper(39) , this study suggests that all three
domains may play direct roles in the membrane translocation step.
EXPERIMENTAL PROCEDURES
Materials
ImmunoPlate MaxiSorp, flat-bottom
96-well microtiter plates were purchased from Nunc. Goat anti-mouse Ig
conjugated to alkaline phosphatase was purchased from Fisher Biotech.
Goat anti-rabbit Ig conjugated to alkaline phosphatase was purchased
from Southern Biochemical Associates (Birmingham, AL). Horse
anti-diphtheria toxin Ig was purchased from Connaught Laboratories
(Swiftwater, PA). Rabbit anti-horse Ig conjugated to alkaline
phosphatase was purchased from Sigma. Gelatin was purchased from Difco.
Sigma 104 phosphatase substrate (5-mg tablets), BCIP, and NBT were
purchased from Sigma. PhastSystem SDS-polyacrylamide gel
electrophoresis gels and supplies were obtained from Pharmacia Biotech
Inc. Nitrocellulose membranes were purchased from Schleicher &
Schuell. Low molecular weight standards were purchased by Electran
(Poole, United Kingdom).
Immunization of Mice
Formalinized diphtheria toxin
was prepared by incubating native toxin with 0.2% (v/v) formalin for 1
week at room temperature (8) or unformalinized free dimer toxin
was prepared by incubating toxin at low pH and 41 °C for 30 min.
The toxin sample was mixed with the adjuvant RAS (Ribi Immunochemicals
Research, Hamilton, MT), and injected into BALB/c mice every 2 weeks
for 2 months. The hybridomas were produced from these mice by the
Tissue Culture/Hybridoma Lab, Microbiology Department, SUNY, Stony
Brook by standard procedures(9) .
Screening Hybridomas
Supernatants from hybridomas
were screened by ELISA and Western blotting. The ELISA protocol used
was similar to that described below, except that the microtiter plate
was incubated at 37 °C and supernatants were not preincubated with
toxin in solution. After preliminary experiments, six hybridomas
(secreting antibodies we named anti-(
)-C1, anti-(
)-C2,
anti-(
)-T1, anti-(
)-T2, anti-(
)-R1, and anti-(
)-R2)
were chosen for further study and then subcloned by limited dilution in
HyClone fetal bovine serum. All except
-C1 were derived from mice
injected with the formalinized toxin.
Antibody Isotyping
Antibody class and subclass
were determined using a mouse-hybridoma subtyping kit purchased from
Boehringer Mannheim as per the manufacturer's instructions.
-C1 antibody was found to be a IgG
, and the other
monoclonal antibodies chosen were IgG
with
light
chains.
Monoclonal Antibody Purification
The antibodies
were purified from supernatants of subclones grown in fetal calf serum
using the QUICKMab affinity resin (Sterogene Bioseparations Inc.,
Arcadia, CA). The supernatants were initially syringe-filtered and
allowed to bind to about 1.5 ml of anti-mouse
affinity resin.
(Fresh resin was used for each antibody to prevent
cross-contamination.) The antibodies were eluted from the column with 2
ml of ActiSep solution and then washed with a pH 4.5 buffer. Fractions
containing antibody as detected by antibody blotting were pooled and
concentrated to about 2 ml, followed by a dialysis against 1 liter of
PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.2
(unless otherwise noted)) overnight at 4 °C. Antibody purity was
determined by SDS-polyacrylamide gel electrophoresis using the
PhastSystem (Pharmacia). The only contaminant appeared to be some
bovine serum albumin (BSA). For the worst cases,
-C1 and
-T2,
there was 20% BSA contaminant, as judged by stain intensity. Antibody
concentration was assayed with the bicinchoninic acid protein assay kit
(Sigma). The approximate antibody concentrations after correction for
BSA were 0.14 mg/ml for
-C1, 0.052 mg/ml for
-C2, 0.25 mg/ml
for
-T1, 0.25 mg/ml for
-T2, 0.12 mg/ml for
-R1, and
0.16 mg/ml for
-R2. (The
-C2 value is only a rough estimate
due to its low concentration.) Antibodies were stored at 4 °C.
Antipeptide
Antibodies
Anti-(
)-C
and
anti-(
)-T
were prepared as described in (10) . They were stored dissolved in PBS, pH 7.1, at a
concentration of 2.75 mg/ml for
-C
and
2.94 mg/ml for
-T
at 4 °C. Aliquots
were diluted to 0.1 mg/ml with PBS, pH 7.5, prior to use.
Diphtheria Toxin
Partially purified diphtheria
toxin was obtained from Connaught Laboratories (Ontario, Canada). Toxin
was further purified as described previously(7, 11) .
Free monomer diphtheria toxin and toxin bound to ApUp was diluted in 5
mM Tris, 1 mM Na
EDTA, 0.02%
NaN
, pH 7.0. The protein concentration of the stock
solutions were determined spectroscopically(6) . Isolated C
domain was prepared as described previously(12) . Isolated T
domain and isolated R domain were kind gifts from the laboratory of
Drs. R. John Collier, Hang-Jun Zhao, and Wei Hai.
Western Blotting Analysis
Free monomer toxin,
isolated T domain, and isolated R domain were diluted to a final
concentration of 0.1 mg/ml into 50 mM Tris-Cl, 1 mM Na
EDTA, 2.5% (w/v) SDS, 0.01% (w/v) bromphenol blue,
pH 8.0, and 7.5% (v/v) 2-mercaptoethanol (loading buffer). 4 µl of
the sample was run on a gradient 10-15% PhastGel, using a
PhastSystem apparatus, and transferred to nitrocellulose using the
transfer accessory. Blots were incubated with purified antibodies, and
after washing, with goat anti-mouse Ig conjugated to alkaline
phosphatase. Color development was performed using NBT and BCIP.
Competition ELISA Protocol
A 96-well microtiter
plate was coated with 1 µg of a 10 µg/ml solution of toxin in
the Lr` conformation in 50 mM carbonate buffer, pH 9.6
(coating buffer), for 1 h at room temperature. (Lr` toxin was prepared
by incubating 80 µg/ml of ApUp-bound monomer toxin in 20 mM acetate, 300 mM NaCl, pH 4.5, at room temperature for 30
min.) Control wells were prepared devoid of toxin. Wells were then
washed at least three times with 200 µl of 25 mM Tris-Cl,
150 mM NaCl, and 2.5 mM KCl, pH 7.2 (washing buffer).
200 µl of a 1% (w/v) gelatin in PBS was added to each well and then
incubated and washed as above. 100 µl of a mixture of PBS-diluted
antibody and toxin preincubated for 2 h was added to each well,
incubated 1 h and washed as above. 100 µl of goat anti-mouse Ig
conjugated to alkaline phosphatase (diluted 1:1000 with 15 mM Tris-Cl, 150 mM NaCl, pH 7.2) was then added, and the
sample was incubated and washed as above. Absorbance at 410 nm minus
490 nm was measured after at least 20 min of incubation with p-nitrophenyl phosphate using a Dynatech MR 600. Under our
experimental conditions, color production was found to be linear in
time, and nearly linear with the concentration of primary antibody
added to the wells. Each sample was prepared in duplicate. The
absorbance of the control well was always found to be negligible when
compared to the absorbance of the sample wells. It should be cautioned
that 50% inhibition of color production may not be equivalent to 50%
inhibition of antibody binding to the wells because of the slight
non-linearity of color with antibody concentration noted above, and the
possibility that some IgGs bind toxin in solution at one antigen
binding site and adhere to the toxin on the wells with the other site.
Dependence of Antibody Binding to Toxin as a Function of
pH
3 µl of 0.57 mg/ml free monomer toxin was incubated with
62 µl of various pH buffers using 20 mM sodium acetate,
300 mM NaCl below pH 5 or 20 mM
Na
HPO
, 300 mM NaCl above pH 5. Sample
pH was reversed to pH 7.1-7.5 with 150 µl of PBS and/or 200
mM Na
HPO
and incubated at room
temperature for 15 min. An aliquot of each sample was added to
PBS-diluted antibody and PBS to give a final volume of 320 µl. The
final toxin concentrations were 2.5 µg/ml for
-C1, 0.75
µg/ml for
-C2, 1.0 µg/ml for
-T1,
-T2, and
-R1, and 1.5 µg/ml for
-R2. The final antibody
concentrations were 0.65 µg/ml for
-C1, 0.98 µg/ml for
-C2, 0.20 µg/ml for
-T1, 0.77 µg/ml for
-T2,
0.56 µg/ml for
-R1, 0.25 µg/ml for
-R2. A competition
ELISA protocol was performed as described above on 100-µl aliquots
of these mixtures. Absorbance values in the presence of toxin divided
by values in the control samples lacking toxin were calculated at each
pH.Similar experiments were performed with anti-peptide antibodies,
with a final toxin concentration of 5.0 µg/ml, and final antibody
concentrations of 4.69 µg/ml for
-C
and 9.38 µg/ml for
-T
. For
antipeptides, absorbance values in the presence of toxin at each pH
divided by the average of the values for the control samples (which
were largely pH-independent) lacking toxin.
Dependence of Toxin Binding to Antibodies on
Temperature
Samples of free monomer toxin (3.5 µl of 0.57
mg/ml) were incubated with 35 µl of 20 mM sodium acetate,
300 mM NaCl, pH 4.4, at various temperatures for 30 min, then
neutralized by the addition of 214 µl of thermally equilibrated PBS
and incubated for an additional 15 min. The samples were then incubated
at room temperature for 5-10 min and diluted to 320 µl with
PBS and antibody as above. The final toxin concentration was 2
µg/ml. The final antibody concentrations were 0.65 µg/ml for
-C1 and 0.20 µg/ml for
-T1. A competition ELISA protocol
was then performed as described above.
Dependence of Binding to Antibodies on Toxin
Concentration
Toxin was prepared in the native(N) and low
pH-treated (Lr` and Lr") conformations. The Lr` and Lr" conformations
were prepared by incubating 2.2 µl of 0.93 mg/ml free monomer toxin
with 35 µl of a 20 mM acetate, 300 mM NaCl, pH
4.4, at 23 °C (Lr` conformation) or 37 °C (Lr" conformation)
for 30 min. Samples were neutralized with 215 µl of PBS at the same
temperature and incubated for 15 min. Native toxin was prepared by
initially mixing 35 µl of 20 mM acetate, 300 mM NaCl, pH 4.4, with 215 µl of PBS and then adding 2.2 µl of
0.93 mg/ml free monomer toxin. Various aliquots of each sample were
then diluted to 100 µl with a 1:5.8 (v/v) mixture of 20
mM, sodium acetate, 300 mM NaCl, pH 4.4, and PBS and
then to 320 µl with PBS and antibody as above. Final antibody
concentrations were the same as in the versus pH section. The
antibody binding was assayed by competition ELISA as described above.
Similar experiments were performed with anti-peptide antibodies, except
that different toxin concentrations and buffer volumes were used. Again
final antibody concentrations were as in the versus pH
section. The concentration of toxin (µg/ml) that yields 50%
inhibition (IC
) in Table 1was obtained from the
graph of percent absorbance of p-nitrophenyl versus concentration of toxin, extrapolating when necessary.
Trypsin Digestion of Native Toxin
10 µl of
1.46 mg/ml free dimer toxin was incubated with 10 µl of a 56
µg/ml fresh trypsin solution (dissolved in PBS) for 17 min at room
temperature. The trypsin-free control contained 10 µl of dimer
toxin and 10 µl of PBS. 2.5 µl of loading buffer was added to a
10-µl aliquot of the reaction mixture or a 10 µl aliquot of the
control sample. 1 µl of each sample was run on a high density SDS
gel using the PhastSystem apparatus and silver-stained or transferred
to a nitrocellulose membrane and developed with
-C1,
-C2, or
-C
.
Antibody Binding to Isolated Domains
The binding
of antibodies to isolated toxin domains was measured using the
competition ELISA (see above).
-C1 binding to isolated C domain,
whole native toxin, and whole toxin in the Lr` conformation was
compared. Isolated native C domain was prepared by mixing 2 µl of
1.29 mg/ml C domain and 249 µl of PBS. The Lr` conformation toxin
was prepared by mixing 3.5 µl of ApUp-bound monomer with 35 µl
of 20 mM sodium acetate, 300 mM NaCl, pH 4.4, for 30
min at room temperature, and then neutralizing with 214 µl of PBS.
The same acetate and PBS were premixed and then added to the toxin to
obtain native toxin. Aliquots were diluted to 120 µl with the same
mix of buffers and then to 320 µl with PBS and antibody as above.
Final antibody concentration was 0.48 µg/ml.
-T
binding to isolated T domain in the
native and Lr` conformation was compared. The Lr` conformation was
prepared by adding 5 µl of 1.4 mg/ml T domain to 87.5 µl of 20
mM sodium acetate, 300 mM NaCl, pH 4.4, for 30 min at
room temperature and then neutralized with 257 µl of PBS. The same
acetate and PBS were premixed and then added to the T domain to obtain
native T domain. Aliquots were diluted to 225 µl with the same mix
of buffers and then to 320 µl with PBS and antibody as above. Final
antibody concentration was 4.7 µg/ml.
-R2 binding to
isolated R domain in the native and Lr` conformation was compared. The
Lr` conformation was prepared by adding 27.5 µl of 0.2 mg/ml R
domain to 110 µl of 20 mM sodium acetate, 300 mM NaCl, pH 4.4, for 30 min at room temperature and then neutralized
with 230 µl of 1:3.6 (v/v) mixture of 200 mM
Na
HPO
and PBS. The same acetate and PBS were
premixed and then added to the R domain to obtain native R domain.
Aliquots of both preparations were then made up to 210 µl with the
same mix of buffers and then diluted to 220 µl with PBS and
antibody as above. Final antibody concentration was 0.045 µg/ml.
RESULTS
Epitope Mapping
Western blotting was used to
epitope map monoclonal antibody binding sites on toxin molecules using
reduced toxin (which gives bands for the A and B chain as well as whole
unnicked toxin), isolated T domain, and isolated R domain (Fig. 1). Three types of antibodies were found: anti-A chain
antibodies, which bound to whole toxin and A chain (C domain); anti-T
domain antibodies, which bound whole toxin, B chain, and isolated T
domain; and anti-R domain antibodies, which bound to whole toxin, B
chain and isolated R domain. Two antibodies against each domain (named
-C1,
-C2,
-T1,
-T2,
-R1, and
-R2) were
chosen for further study. In addition, the binding of the previously
prepared (10) anti-peptide antibodies
-C
and
-T
to toxin was studied. (
)
Figure 1:
Epitope mapping of monoclonal
antibodies. Antibody binding to individual domains was assayed by
Western blotting of SDS-polyacrylamide gels. Lane 1 contains
samples of reduced whole toxin, and has bands corresponding to unnicked
whole toxin, B chain (R+T domains), and A chain (C domain). Lane 2 contains samples of isolated T domain. Lane 3 contains samples of isolated R domain. The polyclonal antibody
blot shows reaction with all of the molecules, whole unnicked toxin, B
chain, and A chain in lane 1; T domain monomers and dimers in lane 2; and R domain monomers and dimers in lane 3.
Antibodies used were:
-C1,
-C2,
-T1,
-T2,
-R1,
-R2, anti-toxin polyclonal.
Higher resolution epitope mapping was performed with
trypsin-cleaved toxin. Incubating native toxin with trypsin results in
the cleavage of the C domain between Lys-39 and Ser-40(12) .
Western blotting of trypsin-cleaved toxin showed monoclonal anti-C
domain antibodies did not bind to the 40-193 fragment (Fig. 2), although as expected
-C
did bind. This suggests part of the epitope for
-C1 and
-C2 lies on residues 1-39, although not all of the epitope
may be within this region.
Figure 2:
Epitope mapping of anti-C domain
antibodies with trypsinized toxin. Trypsinized toxin (lane 1)
and untreated toxin (lane 2) run on a reducing high density
SDS-polyacrylamide gel was stained by silver staining (Ag).
From top to bottom, the lines on the left correspond to the mobility of 16.5-, 14.4-, 8.16-, 6.2-, and
2.5-kDa standards. The positions of whole unnicked toxin, B chain, A
chain, and the 40-193 fragment of the A chain (*) are also shown.
Antibodies
-C1,
-C2, and
-C
binding to reduced trypsinized toxin run on a high density gel
was performed by Western blotting. High density gels expand slightly
during transfer, which causes slight shifts in bands location relative
to silver stain positions.
Epitopes were also mapped with cysteine
cleavage reagent 2-nitro-5-thiocyanobenzoic acid. Antibody binding to
cleavage products of mutant toxins in which a single cysteine was
substituted for either Glu-162 or Ser-337 was examined. Anti-C
antibodies bound to the 1-161 fragment, but not to the
162-535 fragment, and anti-T domain antibodies bound to the
1-336 fragment but not the 337-565 fragment (data not
shown). This suggests that the anti-C domain antibodies bind between
residues 1 and 161, and the anti-T domain antibodies bind between
residues 194 and 336.
Additional data on epitope locations comes
from the results of antibody binding to octapeptides that cover the
entire sequence of the B chain (10) . The
-R1 antibody
binding profile shows an affinity for octapeptides with sequences
identical to the loop region between the T and R domain and residues
486-500 (RB8) (data not shown). The binding profile of
-R2,
together with hydroxylamine cleavage patterns (data not shown),
suggests
-R2 binds residues 454-465 (RB6).
The Effect of pH-induced Changes in Toxin Conformation on
Antibody Binding: Competition ELISA
Diphtheria toxin undergoes a
conformational change at pH 5.3 that plays a critical role in its entry
into cells (4) (see Introduction). Above pH 5 the toxin remains
in the native(N) state, but after exposure to low pH at 23 °C, the
toxin undergoes a partial unfolding process that results in its taking
on a hydrophobic, membrane-inserting
conformation(5, 6, 7, 13, 14, 15, 16, 17, 18, 19, 20) .
In this conformation, the B chain has undergone a partial unfolding
process but the C domain remains folded(7) . In order to
further characterize the changes that the toxin undergoes at low pH,
the pH dependence of antibody binding to toxin was studied. As with
many antibodies, anti-toxin binding at low pH was found to be too weak
to measure. Therefore, binding was examined after low pH was reversed
to neutral. Under these conditions previous studies have shown the
toxin maintains the structural changes that occur at low
pH(7) . (This low pH-treated state is called the Lr`
conformation.
)To measure the dependence of antibody
binding on the pH at which toxin is incubated, toxin was preincubated
at various pH values in solution and its antibody binding measured by a
competition ELISA after pH neutralization. In this assay the amount of
antibody binding to the toxin in solution is measured by its inhibition
of antibody binding to toxin-coated wells(21) . The amount of
antibody bound to the wells is assayed by the amount of p-nitrophenyl absorbance generated after incubation of the
wells with an alkaline phosphate conjugated to an anti-mouse or
anti-rabbit Ig and addition of p-nitrophenyl phosphate. The
more antibody bound to the toxin in solution, the less binds to the
wells, and the lower the absorbance value measured.
The
degree of inhibition of antibody binding to ELISA plates by toxin in
solution was quantitated through the decrease in ELISA absorbance in
the presence of toxin relative to the controls lacking toxin.
The Effect of pH-induced Changes in Toxin Conformation on
Antibody Binding: Anti-C Domain Antibodies
Fig. 3A illustrates effect of the pH at which toxin is incubated upon
anti-C binding.
-C1,
-C2, and
-C
behave similarly in that their binding is stronger to low
pH-treated toxin, with a sharp transition in binding near pH 5, the
value at which the low pH-induced change in toxin conformation has been
shown to occur(5, 6, 20) . Antibody binding
to toxin can be quantified by the comparison of the concentration of
toxin sufficient to inhibit the ELISA color reaction by 50%
(IC
) (Fig. 4, A and B, and
5A). The IC
values derived from these experiments
are given in Table 1and show the difference in binding to low
pH-treated (Lr` conformation) and native toxin is greatest in the case
of
-C1 (about 30-fold), but is also significant for the other
anti-C antibodies (about 2.5-fold).
Figure 3:
Effect of pH on antibody binding to toxin
assayed by competition ELISA. The inhibition of antibody binding to
ELISA plates obtained by preincubating antibodies with toxin (treated
at the appropriate pH and 23 °C) in solution was measured through
the decrease in absorbance. The absorbance relative to that in the
absence of toxin in solution (defined as 100%) is shown. The antibodies
used were as follows: A,
-C1 (
),
-C2 (
),
and
-C
(+); B,
-T1
(
),
-T2 (+), and
-T
(
); and C,
-R1 (+) and
-R2 (
).
Points shown in Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7are
the average of duplicate samples. The absorbance values of duplicates
were generally within 10% of each other.
Figure 4:
Assay of monoclonal antibody binding to
the native(N) and low pH-treated (Lr`) conformations of toxin using
competition ELISA. The inhibition of antibody binding to ELISA plates
obtained by preincubating antibody and N (
) or Lr` toxin (
)
in solution was measured through the decrease in absorbance. Antibodies
used were as follows: A,
-C1; B,
-C2; C,
-T1; D,
-T2; E,
-R1; F,
-R2.
Figure 5:
Assay of antipeptide antibody binding to
the native(N) and low pH-treated (Lr`) conformation of toxin using
competition ELISA. Samples contained N (
) or Lr` (
) toxin.
See Fig. 4for details. Antibodies used were as follows: A,
-C
; B,
-T
.
Figure 6:
Assay of antibody binding to isolated
toxin domains using competition ELISA. Panel A contains native
whole toxin (
), Lr` whole toxin (
), and isolated native C
domain (+). Panel B contains isolated native (
) and
Lr` (
) T domain. Panel C contains isolated native
(
) and Lr` (
) R domain. See Fig. 4for details.
Antibodies used were: A,
-C1; B,
-T
; and C,
-R2.
Figure 7:
Assay of antibody binding to toxin
incubated at various temperatures using competition ELISA. The
inhibition of antibody binding to ELISA plates obtained by
preincubating antibodies with toxin (treated at pH 4.5 and various
temperatures) in solution was measured through the decrease in
absorbance. The absorbance relative to that in the absence of toxin in
solution (defined as 100%) is shown. The antibodies used were
-C1
(
), and
-T1 (
).
The Effect of pH-induced Changes in Toxin Conformation on
Antibody Binding: Anti-T Domain Antibodies
Similar experiments
were performed with anti-T domain antibodies. Fig. 3B illustrates the effect of pH at which toxin is incubated upon
anti-T binding.
-T1,
-T2, and
-T
each react differently with native toxin and low pH-treated
toxin.
-T1 shows no significant dependence on the pH at which
toxin was preincubated. This is confirmed by the observation that
IC
s for native and low pH-treated (Lr`) toxin binding by
-T1 are the same (Fig. 4C and Table 1).
-T2 antibody binds to native toxin more strongly than low
pH-treated toxin with about a 7-fold preference for native toxin (Fig. 4D and Table 1). Again, there is a
transition at pH 5 between the strongly and weakly bound toxin
conformations. In contrast,
-T
binds to
the low pH-treated toxin more strongly (13-fold) than to native toxin (Fig. 5B and Table 1), although again with a
transition at pH 5 (Fig. 3B).
The Effect of pH-induced Changes in Toxin Conformation on
Antibody Binding: Anti-R Domain Antibodies
Fig. 3C illustrates the effect of pH at which toxin is incubated upon
anti-R binding. pH has an opposite effect on
-R1 and
-R2
binding to toxin.
-R1 binds native toxin more strongly than low
pH-treated (Lr`) toxin (Fig. 4E), with a 5-fold
preference for native toxin (Table 1).
-R2 binds low
pH-treated toxin more strongly than native toxin (Fig. 4F), with a 2.2-fold preference for the former (Table 1).
The Effect of pH-induced Changes in Toxin Conformation on
Antibody Binding: Binding to Isolated Domains
Binding of several
antibodies to isolated domains was also measured. Isolated T and R
domains appear to take on native and low pH-induced conformations
similar to those they have in whole
toxin(22, 23) .)
Therefore, antibody
binding to native and low pH-treated domains could be studied. The
isolated domain binding of
-T and
-R antibodies that prefer
binding the low pH-treated whole toxin was measured to see if the
increased binding at low pH is due to increased exposure of their
epitopes due to a loss of interdomain interactions, or due to changes
within the domain. Fig. 6shows
-T
binding to isolated T domain and
-R2 binding to the R domain
have the preferential binding to low pH-treated protein seen in whole
toxin, supporting the latter proposal.Isolated C domain takes on a
conformation similar to that in whole toxin (24, 25) and undergoes only reversible changes at low
pH, and thus can only be studied in the native state.
-C1 binding
to isolated native C domain was also measured and found to be much more
similar to the strong binding of C domain in low pH-treated whole toxin
rather than the weaker binding of C domain in native whole toxin (Fig. 6).
The Effect of Thermally Induced Conformational Changes on
Antibody Binding to Toxin
Above 26 °C at low pH (4.5)
diphtheria toxin undergoes a change in conformation to a structure in
which, in addition to the partial unfolding seen in the B chain at low
pH, the C domain partly unfolds(7) . After pH neutralization,
this conformation is called the Lr" conformation. The relative role in
translocation of the Lr" toxin and the above-studied Lr` toxin, which
predominates at lower temperatures, remains unresolved(26) .
Therefore, it was of interest to examine the difference between these
conformations by antibody binding.The competition ELISA was used to
examine the binding of
-C1 and
-T1 to toxin preincubated at
various temperatures (Fig. 7). A transition is seen close to 26
°C for both antibodies, showing that antibody binding responds to
the Lr` to Lr" conformational change.
An ELISA competition assay was
then performed with all of the antibodies using toxin in the Lr"
conformation. The IC
values obtained are given in Table 1and show that all the antibodies reacted 2-9-fold
more strongly with the Lr` toxin than with the Lr" toxin. We attribute
this partly to steric blocking arising from aggregation of the toxin
hiding epitopes in the Lr" conformation (see ``Discussion'').
DISCUSSION
Using Monoclonal Antibodies to Evaluate Diphtheria
Toxin Structure
Binding of anti-diphtheria toxin monoclonal
antibodies to the toxin has previously been studied in other
laboratories(27, 28, 29, 30, 31, 32, 33) which have found that monoclonal antibodies that bind to
various sites on the toxin can be obtained. The studies of Zucker and
Murphy(29, 30) tried to designate function to a
particular area of the toxin by examining which functions were blocked
by antibodies, but did not examine the effect of pH on toxin behavior. A study by Rolf and Eidels (33) did identify some
monoclonals that bind to low pH-treated toxin, but the number of pH
values chosen for binding studies were insufficient to determine
whether the pH-dependent change in binding they observed corresponded
to the conformational transition at pH 5. Furthermore, the dependence
of binding on pH was weak, perhaps reflecting the sensitivity of the
immunoprecipitation method used.
In this study, antibodies to each
domain of the toxin have been identified. These studies show that the
binding of monoclonal and antipeptide antibodies can respond strongly
to conformational changes in diphtheria toxin. Such antibodies should
be useful tools for establishing whether toxin mutants fold and undergo
conformational changes similar to those in whole toxin.
Just as
important, these studies provide some new insights into the mechanism
of diphtheria toxin insertion into membranes. One central observation
in this study is that changes in all three domains occur at low pH. One
obvious question is what changes are occurring in each of the domains?
This is complicated to answer because several factors can influence
antibody binding. A difference in antibody binding to two conformations
could result from a difference in exposure of an epitope, or a change
of an epitope into a conformation that is more weakly or tightly bound.
Furthermore, a change in exposure of an epitope could be due to a
change in the position or conformation of a sequence of a neighboring
domain, or intermolecular aggregation.
Low pH-induced Changes in C, T, and R Domain Structure in
the Lr` Conformation
Despite these difficulties, the changes in
antibody binding to toxin do provide some interesting details to what
is occurring at low pH. Let us first consider the behavior of the toxin
in the Lr` conformation. Previous studies have shown that although
there is some partial unfolding of the B chain in this conformation the
C domain (A chain) remains folded(7) . Therefore, the increased
binding to low pH-treated toxin found for all three anti-C antibodies
examined indicates there is increased exposure of the epitopes on the C
domain to solution after low pH treatment. This suggests that the
degree of contact between the C domain and the B chain (T plus R
domains) must decrease at low pH. A related possibility is that the C
domain undergoes a small conformational change that promotes antibody
binding. In fact, a small conformational change in residues 66-78
and 169-176 has been seen in isolated C domain relative to whole
toxin by crystallography(25) , and is believed to be due to the
loss of C domain interactions with the T domain. It is possible that
our monoclonal antibodies bind C domain in whole toxin exposed to low
pH more tightly due to a similar loss of C domain-T domain
interactions. However, a more general increase in exposure at low pH is
also very likely because the
-C
, which
does not involve the residues that change structure upon loss of T
domain interaction, also binds more tightly at low pH. This conclusion
is consistent with evidence that the interactions between the C and R
domain are lost at low pH(34) .Additional changes occur in
the T and R domain. The observation that the differences between the
binding of some antibodies to these domains in the native and low
pH-treated states are seen with isolated domains as well as whole toxin
indicate that they are not solely due to changes in interdomain
interactions, and probably reflect conformational changes within each
of these domains at low pH. It is likely that there are conformational
changes in these domains due to partial unfolding and exposing
hydrophobic surfaces at low pH(12, 22) . This
conformational change would decrease the recognition to low pH-treated
toxin of antibodies such as
-R1 because the epitope would become
hidden or change conformation. The same conformational change could
cause increase antibody binding to low pH-treated toxin for antibodies
such as
-T
and
-R2 due to increased
exposure or due to cooperative binding to low pH induced toxin
aligomers (although see below).
Toxin Behavior in the Lr" Conformation
For all of
the antibodies tested, it appeared that binding to toxin in the Lr"
conformation was less than binding to the Lr` conformation. It is
likely that in solution strong aggregation of the toxin in the Lr"
conformation generally decreases antibody binding to this conformation
of the toxin. Previous studies have shown that the hydrophobic behavior
of diphtheria toxin at low pH induces its aggregation in
solution(6, 35) . The observation that membrane
insertion, which should eliminate the presence of aggregates held
together by hydrophobic interactions, greatly increases antibody
binding to the Lr" conformation (see accompanying paper(39) )
supports the proposal that aggregation in solution influences antibody
binding. In contrast, aggregation does not seem to be a major influence
on antibody binding to toxin in the Lr` conformation because the
antibody binding to Lr` toxin in solution and inserted in model
membranes is generally similar (compare Table 1of the present
report to Table II in (39) ). (
)
A Model for the Changes in Diphtheria Toxin
at Low pH and Implications for Membrane Insertion
Based on the
changes observed in diphtheria toxin at low pH, a schematic model
summarizing the changes in toxin structure leading to membrane
insertion can be proposed. The first stage would involve the loss of
interactions between C and R domains, proposed by Bennett and
Eisenberg(34) . The next step would involve the major
conformational changes observed in both the T and R domain, greatly
increasing their hydrophobicity (Fig. 8). These conformational
changes suggest that interactions between all three domains would break
or become much weaker. This would also result in sites on all three
domains becoming exposed to the aqueous environment after treatment
with low pH, which could aid in membrane insertion. The unfolding of
the C domain into a hydrophobic conformation, which requires slightly
more extreme conditions, may also occur at this time. Thus a number of
coordinated changes may play a role in the membrane insertion process.
Figure 8:
Schematic figure of diphtheria toxin
conformational changes at low pH. A change in conformation is shown by
a change in outline shape. The figure shows that in the low pH-treated
Lr` conformation, there is an increase in exposure of sites to solution
as well as a change in T and R domain conformations. A small
conformational change seen in isolated C domain may also occur (25) . In the Lr" conformation, an additional unfolding of the
C domain conformation occurs, as shown in previous
studies(7) .
The observation of low pH-induced conformational changes in the R
domain is important because it implies that the R domain plays an
important role in insertion, rather than just functioning in binding to
the cellular receptor(36, 37, 38) . This is
reinforced by the recent observation that the R domain becomes
hydrophobic at low pH(23) , and the results in the accompanying
report, showing that the R domain inserts into model membranes in a
fashion such that some sites translocate across the bilayer.