The
ɛ2ζ2
Antitoxin/Toxin
System
Danny Iwamoto '10 and Basil Kahwash '10
Contents:
I. Introduction
The low-copy-number pSM19035
plasmid, isolated from the pathogenic bacterium Streptococcus
pyogenes,
contains genes for erythromycin resistance, as well as the
ω-ε-ζ (omega-epsilon-zeta)
operon
in its SegB region, which is responsible for encoding the
ε-ζ antitoxin/toxin
system {Reference 3}. This functions as a postsegregational killing
(PSK) system that
induces
programmed cell death (PCD) if the pSM19035 plasmid is ever
unsuccessfully
copied into each daughter cell during cell division. This process is
illustrated in this
pop-up
diagram
. This
ensures the plasmid is inherited and
maintained through subsequent generations of the prokaryote, otherwise
resulting in cell death {2}. This
is also
known as a plasmid addiction system (PAS), since any newly born
cells lacking the
plasmid will die {3).
The gene
encodes for the unstable antitoxin ε and
the stable toxin ζ proteins. Proteases
are thought to be responsible for the eventual breakdown of each
protein,
though the ζ toxin has been shown to significantly outlast the
ε antitoxin in the cytoplasm {1}.
A stable heterotetramer is formed by the binding of two toxin proteins
and two
antitoxin proteins, forming a harmless and inactive complex in the cell
cytosol. By continuously and excessively
producing antitoxin, the system's toxic effects are counteracted as
long as the plasmid
is maintained. Loss of the plasmid, damage
of the antitoxin gene, or unsuccessful copying into a new daughter cell
results
in decreasing amounts of antitoxin and the liberation of toxin proteins
in the
cytoplasm which then become active and inhibit cell growth, cause
filamentation, and eventually induce PCD {2,3}.
II. General Structure
The complete inactivation complex
is
a heterotetramer composed of 754 amino acids. The
complex consists of two
ζ proteins and two ε proteins
,
which form a dumbbell-shaped structure so that the
two ζ proteins are bound to opposite sides of the ε2
dimer {2}.
The ε protein contains 90
amino acids
,
which form three
alpha helices
,
interacting as a coiled coil and protecting
the majority of the non-polar
residues by internalizing them within the
protein
through
hydrophilic reactions.
Two ε proteins form the ε2 dimer
,
which further protects non-polar residues by
sandwiching them between the
ε proteins
{2}.
The
ζ protein contains 287 amino
acids
,
arranged into 11
alpha helices
and 6 beta sheets
.
The beta sheets are centrally located within the
ζ protein, and all
are parallel, except for the antiparallel
final sheet
.
The two C-terminal helices are
not strongly bound to the main body of the protein, forming an exposed helix-loop-helix
appendage
,
though it does not seem to serve a function in the
toxicity of the protein. The ζ
protein also contains a phosphate-binding
loop (or P-loop),
an important
region for the toxin's function
...
{2}
III. The Functions of the Zeta and Epsilon
Proteins
The ζ protein is the toxic protein of
the pair, although the exact toxic mechanism of the
protein is unknown. The toxin, however, does
contain a Walker
ATP/GTP binding motif
,
also known as a P-Loop, indicating that the
ζ protein exhibits similar folding patterns to
phosphotransferases, suggesting a similar function. However, the
substrate for this binding site is unknown, though ATP is
involved as the phosphoryl group donor. Thus, the ζ
protein probably acts as a phosphotransferase to remove a single
phosphoryl group from ATP, and bind it to the substrate; this is
the ζ protein's toxic
activity {2}.
The ε
protein, as the antitoxin protein, therefore blocks this binding site
sterically by forming the inactivation complex heterotetramer
{2}.
Studies have
shown in vivo
and in vitro
stability for the ζ protein as over 60 minutes, but
the ε
protein is stable
for less than 18 minutes {1}.
Therefore, the
ε antitoxin must be produced often in order to
counteract the ζ
toxin. If, during cell replication, the pSM19035 plasmid is not carried
by a daughter cell, proteases will degrade the existing toxin and
antitoxin proteins. However, the antitoxin proteins will be degraded
over three times as quickly, resulting in a stoichiometric excess of
toxin proteins, which then begin to induce PCD {1,2}.
IV. The Zeta
Activity Sites
The ζ
protein contains two binding sites: one
for binding ATP
,
and the
other
for binding
the unknown substrate
. These
sites are
close in proximity, since one of ATP’s phosphoryl groups must
be transferred to
the substrate
.
ATP and
the substrate and held in place by a number of stabilizing reactions
involving the residues of the ζ protein {2}.
ATP,
once it
enters the ζ protein, is held in place by the P-loop:
residues
40-47 of the ζ
protein
.
Lys-46
in particular
of
the P-loop plays a major
role by
forming hydrogen bonds to one oxygen atom each of ATP’s
β- and γ-phosphates. In
addition, other nearby residues such as Arg-158 and Arg-171
bend
towards
ATP to stabilize it, also by forming hydrogen bonds to ATP’s
phosphoryl groups {2}.
Once
ATP is
bound, the ζ protein is opened into a conformation so that the
substrate can
bind. Since the substrate’s molecular structure is unknown,
it is unclear as to
what particular residues play important roles in stabilizing the
substrate.
However, assuming no dramatic conformation changes occur upon substrate
binding, the substrate must be a small molecule, or a solvent exposed
segment of a protein or nucleic acid {2}. The
substrate is likely to be held near the ATP binding site,
between a
beta
sheet and two alpha helices of the ζ protein
,
a
region also filled with numerous
water molecules which aid
in ATP hydrolysis. The Mg2+ ion is an
important
atom also involved in ATP-hydrolysis, though this atom could not be
identified in
electron density mapping of the complex {2}. It is assumed that Glu-116
binds
this catalytically important ion, which neutralizes the developing
negative
charge during ATP hydrolysis. Meanwhile, Asp-67
deprotonates
the
substrate, which allows the substrate to perform a nucleophilic attack
on the
γ-phosphate of ATP. Lys-46,
Arg-158
and Arg-171, of the ATP binding region,
help
stabilize the resultant pentacovalent
transition
state of the γ-phosphate through hydrogen bonding to the
β- and
γ-phosphates. Collapse of the
pentacovalent transition state yields the phosphorylated substrate and
ADP, completing the phosphotransfer {2}.
V. Inactivation
Complex
The stable inactivation complex
heterotetramer is formed when two toxic ζ proteins bind to the
ε2 dimer
. The
fact that ε is unstable means that ε
will almost always be found in the
ε2ζ2 complex
{2}.
Bonding between ε and
ζ occurs in such a way that ATP is
prevented from binding
to ζ, also effectively preventing the substrate from binding
to ζ.
Most toxin-antitoxin interactions involve a
single helix of ε {2}
.
Residues
on the N-terminal of this
helix make
contacts
with the residues of the ζ
protein
.
For example, the
carboxylate groups of Glu-12
and
Glu-16 of ε form hydrogen
bonds to
the amino group of Arg-158
of ζ, pulling it away from where it is needed in the ATP
binding site. Other
residues on ε serve to sterically impede the
binding of ATP to
ζ, such as Tyr-5
and Phe-9
,
which block the ATP binding site of
ζ (the P-loop,
Lys-46,
Arg-158,
and Arg-171)
{2}. As a result, the ζ protein is inactivated
through steric hindrance by the ε upon formation
of
the stable inactivation complex heterotetramer
.
This final button shows the complete inactivated heterotetramer, and
the key amino acid residues involved.
VI. References
1.
Camacho, A. G., R Misselwitz, J. Behlke,
S. Ayora, K. Welfle, A. Meinhart, B. Lara, W. Saenger, H. Welfle, and
J.C. Alonso. 2002. In vitro and in vivo stability of the epsilon2zeta2
protein complex of the borad host-range Streptococcus pyogenes pSM19035
addiction system. Biol.
Chem. 383:1701-1713.
2. Meinhart,
Anton, Juan C. Alonso, Norbert Sträter, and Wolfram Saenger.
2003. Crystal structure of the plasmid maintenance system
ε/ζ: Functional mechanism of toxin ζ and
inactivation by ε2ζ2 complex formation. PNAS
100:1661-1666.
3. Zielenkiewicz, Urszula and Piotr Cegłowski. 2005. The
Toxin-Antitoxin System of the Streptococcal Plasmid pSM19035. Journal
of Bacteriology 187:6094-6105.
Back
to Top