E.
coli Shiga 2 Toxin
Rachel Martin '11 and Daniel Riggins '12
Contents:
I.
Introduction
Stx, or Shiga toxin,
is a protein
produced by the
bacterium Shigella
dysenteriae, and is named for
Kiyoshi Shiga, who first
discovered the microorganisms behind dysentery {1}. A variation of this
toxin, Stx2,
is produced not by S.
dysenteriae,
but by Escherichia
coli (most notably
by strain O157:H7), and is one of the main sources of food poisoning
{2}. Though
another variation of the Shiga toxin, Stx1, is also produced by E. coli,
patients infected with the
latter and Stx are much less likely to develop serious illness than
those
infected by the former; thus, differences in toxin structures are of
great
interest to the public health community {3}. While Stx and
Stx1 are nearly identical, the Stx2 protein structure contains
some
major
alterations that augment its pathological efficiency.
Shiga toxins bind
to receptors on the
outside of the cell and are taken up into the endoplasmic reticula (ER)
{4}. Because
the receptors are not found in meat animals like cattle and swine, they
can
harbor toxic bacteria with little consequence; however, when the toxins
are
ingested by humans, they quickly enter the cells {2}. Once inside, Stx
proteins
act as N-glycosidases,
depurinating
specific adenines in the 28S
rRNA of
the 60S subunit of ribosomes.
Cleavage
of this adenine base leads to the incapacitation of the ribosome and
the
termination of protein synthesis {4}.
II.
General Structure
Like
other members of its toxin family, Stx2 is made up of one alpha subunit
and five beta subunits.
The alpha
subunit contains the
catalytic activity (found in A1) that cleaves adenine from the ribosome.
The active site's main components are arginine 170,
valines 78 / 162, serine 112, alanine 166, and tyrosines 77 / 114
{4}.
The pentameric
ring formed by the beta
subunits binds to the membranes of
target cells. All stx toxins preferentially bind to
glycosphingolipids via fifteen active
sites (three per beta monomer)
{5}.
The
carboxy-terminal portion (A2)
of the alpha
subunit links it to the
pentameric
ring by extending through its center
pore. A2
traverses through it starting as an alpha helix,
then in an extended conformation. A2
ends as a terminal helix that projects
out of the B-pentamer
at 30 degrees {5}.
A1
is
linked to A2
via the polypeptide chain and a disulfide
bond between
the sulphur
atoms of
cysteine 241 and cysteine 260.
In
vivo, A1
separates from the rest of the toxin by nicking the
polypeptide chain and reducing a disulfide bond. This
structure shows the polypeptide
chain already broken.
Henceforth after breaking, A1
is active as an N-glycosidase. Unlike in the structure of Stx,
the
active site is accessible to small molecules before A1
parts from A2
{5}.
III.
Adenine Binding
When
the toxin crystals are grown in the presence of adenine or adenosine,
the ligand bound in the final product is always adenine. This
suggests that the enzyme is active as an
N-glycosidase, that is, it cleaves via hydrolytic attack the purine
base from a sugar on RNA
at the site of the glycosidic bond {4}.
When
bound in the active site, the adenine has
hydrogen bonding
interactions with Val-78,
Ser-112,
and Arg-170.
Additional interactions via Van der Waals
forces come
from Val-162,
Ala-166,
Tyr-77,
and Tyr-114
{4}.
When
adenosine or adenine enters the active site,
it displaces molecules of
formic acid and water. While the adenine aligns, Tyr-77
rotates slightly such that
it
ring stacks with adenine's ring {4}.
Only
the alpha subunit is
displayed in complex with the adenine
because
it is unlikely that such a large
molecule would be able to approach the 28s rRNA in order to
bind.
The catalytic site is not active until after A1
separates from the rest of the holoenzyme
{4}.
Although
the mechanism of catalysis has not been formally determined for the
Stx2 enzyme, it is predicted from amino acid sequence alignments that
Stx2's mechanism is highly analogous to that of Ricin. Ricin
is
heterodimeric toxin derived from Ricinus
communis
(the
Castor Bean) {5}.
The
adenosine (shown here as adenine) base binds into a pocket of the Ricin
active site between Tyr-80
and Tyr-123,
stacking against
their rings. As with
Stx2, Tyr-80
must rotate
~45 degrees upon the base entering.
Arg-180
acts as an
acid to partially protonate N3 on the
Adenine. Glu-177
stabilizes the transition state or serves as
a base to activate a water
molecule
that attacks N9
where the glycosidic bond would be located were this an adenosine
molecule {6}.
IV.
Mechanism of Entry into the Cell
Shiga
toxins enter the cell by binding with the globotriaosylceramide
receptor (Galα1-4Galβ1-4-glucosyl ceramide, or Gb3)
through the B pentamer {4}. (A variation of Stx2, Stx2e, binds
selectively to the globotetraosylceramide receptor, Gb4, allowing it to
target different cells {5}.) The toxin is then taken through
clarthrin-coated cavities and taken up by the endoplasmic reticula (ER)
via retrograde transport. In the ER, the polypeptide chain between A1
and A2 is nicked, and the disulfide bond connecting the two portions is
reduced, releasing A1 into the cytoplasm where it begins its activity
as an N-glycosidase {4}.
There
are three main
binding regions on each beta
monomer.
The first region is located in the groove between two
beta
subunits where they
cooperatively form a seven-stranded beta sheet.
The site is comprised of asparagine
14,
aspartate 16, threonine 20, glutamate 27, tryptophan 29, and
phenylalanine 30.
The indole ring of Trp-29
stacks against the phenyl ring of 3-(1-pyridinio)-1-propanesulfonate
(PPS),
which mimics the binding of the second
galactose ring of the carbohydrate receptor. The formic acid
partially
imitates binding of the first galactose ring {5}.
Region two is made up of alanines
1 / 63, serine
54, and cysteines
3 /
56. In order for
Gb3 to bind, it is thought that a disulfide
bond between the cysteine must change conformation {5}.
Region three is found at the base of the protein where the alpha
subunit exits the pore formed
by the beta subunits.
Because of proximal placement, all five
sites for region three must move in concert with each other in order
for Gb3 molecules to bind. Each binding region consists of tryptophan
33 and asparagine 34.
It is thought that packing of
the last few residues of the alpha
subunit against the tryptophan
residues on beta subunits
may interfere with binding in some cases {5}.
V.
Differences From Stx and Stx1
Though
Stx/Stx1 and Stx2 are fairly homologous (56% homology in the A subunit,
64% in B), Shiga toxin and Type I Shiga toxin have been associated with
much milder forms of disease than Type II {4}. Until structures of the
molecules were determined, the reason for these observations was not
immediately clear; comparison of the crystal structures of these
proteins gives insight into this phenomenon.
The
accessibility of the active site is the most prominent difference
between Stx/Stx1 and Stx2. The polypeptide chain linking A1
and A2
lies
near the active site; in the Stx protein, A2
residues obstruct this
adenine-binding site, decreasing the efficiency of depurination
{5}.
Interactions between the carboxyl
terminus
of the A
subunit and the
B subunits (not shown) in Stx2 also contribute
to differences in the
proteins’ efficacies: the carboxyl
terminus of the A subunit
of the Stx2 protein threads through the B pentamer and forms a short
α-helix, whereas the same stretch
of polypeptide is
disordered in Stx/Stx1.
This Stx2 A subunit carboxy tail has been shown
to enhance the toxicity of a subtype of Stx2, Stx2d-activatable, in the
presence of elastase, found in mucus. Differences in B pentamer binding
sites may also contribute to disparities between Stx and Stx2, as the
binding
sites mediate B attachment to cell receptors and thus
efficiency of cell entry by the toxin {5}.
VI.
References
1. Trofa, Andrew F., Hannah
Ueno-Olsen, Ruiko Oiwa, Masanosuke Yoshikawa. Dr. Kiyoshi Shiga:
Discoverer of the Dysentery Bacillus. 1999. Clinical Infectious
Diseases 29(5): 1303-1306.
2. Bower JR. Foodborne diseases: shiga toxin producing E. coli (STEC).
1999. J Pediatr Infect Dis 18: 909-10.
3. Friedrich, Alexander W., Martina Bielaszewska, Wen-Lan Zhang,
Matthias Pulz, Thorsten Kuczius, Andrea Ammon and Helge Karch.
Escherichia coli Harboring Shiga Toxin 2 Gene Variants: Frequency and
Association with Clinical Symptoms. 2002. The Journal of Infectious
Diseases 85(1): 74-84.
4. Fraser, Marie E., Maia M. Cherney, Paola Marcato, George L, Mulvey,
Glen D. Armstrong, and Michael N. G. James. Binding of adenine to Stx2,
the protein toxin from Escherichia coli O157:H7. 2006. Acta
Crystallographica F62: 627-630.
5. Fraser, Marie E., Masao Fujinaga, Maia M. Cherney, Angela R.
Melton-Celsa, Edda M. Twiddy, Alison D. O'Brien, and Michael N. G.
James. Structure of Shiga Toxin Type 2 (Stx2) from Escherichia coli
O157:H7. 2004. The Journal of Biological Chemistry F62: 627-630.
6. Miller, Darcie J., Kabyadi Ravikumar, Huafeng Shen, Jung-Keun Suh,
Sean M. Kerwin, and Jon D. Robertus.. Structure-Based Design
and
Characterization of Novel Platforms for Ricin and Shiga Toxin
Inhibition. 2002. J. Med. Chem 45: 90-98.
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