Bacillus
subtilis is a
non-toxic bacterium commonly found in soil, usually in the form of a dormant
spore. It is extremely hardy due to a
remarkable list of adaptations to environmental threats. These include the production of antibiotics
and degrading enzymes, amalgamation into biofilms,
the formation of an endospore and
even the destruction of sibling bacteria.
This impressive
range of threat responses is triggered by many different adaptation genes, which
are governed by an army of regulators, some specific and some global.
One notable
universal regulator protein is AbrB, which represses several adaptation genes
when cell conditions are favourable. There
are two mechanisms that block this repressor, when the bacterium faces a
threat.
Under stress, an
upstream regulator, SpoOA, is phosphorylated and, in an envy inducing display
of multi-tasking, is able to bind to and inhibit the gene for AbrB and also
activate the gene for anti-repressor AbbA.
AbbA binds to
the repressor AbrB and blocks it, thereby allowing adaptation to environmental
threat. AbbA has recently been revealed
as a DNA mimic, which competes very effectively for the AbrB binding site by
copying key DNA characteristics.
Inspection of
the primary structure of AbbA reveals 65 residues of which 20 are polar. Given that the core of a protein tends to be
hydrophobic, this implies an polar surface.
The next step
was to establish the oligomerisation of the protein in its native state. Size-exclusion liquid chromatography (SELC)
and native mass
spectrometry both showed that the natural size of AbbA was double the mass
of the monomer, that is, it dimerises.
NMR was used to
solve the dimer structure.
This figure
shows the ten lowest energy structures of the AbbA dimer obtained using
NMR. One monomer has helices coloured
blue to green whilst the second is yellow to red. (PDB 2LZF).
Figure taken
from Tucker, A.T. et al (2014)
The monomer
consists of three alpha helices, connected by two loops with a fairly
unstructured N terminus. In the dimer
there are substantial interactions between helices two and three of each
monomer, which are largely hydrophobic, and further hydrophobic interactions
between helices one and two.
To determine the
binding site for AbbA and AbrB, SELC studies were performed on AbbA with the C
terminus of AbrB and then with the N terminus.
The team found that AbbA binds only to the N terminus, the region of the
protein that is responsible for binding to DNA.
Previous mutation
studies had shown that four arginine residues are responsible for AbrB binding
to DNA, namely R8, R15, R23 and R24.
Each of these was mutated to observe the impact on AbbA binding and
three of them, R8, R15 and R23, were found to be critical, potentially
indicating a similar binding mechanism.
To determine the
strength of the competition offered to AbrB-DNA binding by AbrB-AbbA binding, isothermal
titration calorimetry was used to measure the dissociation
constants. This technique is covered
in TSMB, one of the courses available after PPS. The dissociation constants showed that the
binding strengths of the two pairs of molecules were similar, such that AbbA
offers a significant threat to AbrB-DNA binding.
The last stage
of the investigation was to determine the interaction between the Abba
homodimer and the AbrB homodimer.
Molecular docking was
used to model this interaction and showed a sizeable interaction area with a
complex pattern of 18 hydrogen bonds and 16 salt bridges. This site is at the highly negatively charged
terminal of AbbA and the DNA binding face of N terminal AbrB.
The first helix
of each AbbA monomer has to pull back to allow this strong interaction but
AbbA's second and third helices maintain their conformation and act as a
stabilising anchor.
The picture
below gives a striking illustration of the extent of AbbA's mimicry of
DNA.
This figure
illustrates the similarities between AbbA and the DNA phosphate backbone. (a) is the NMR structure of AbbA with
positively charged residues shown in blue and negatively charged in red. This can be compared with (c), a DNA fragment
(PDB 1BNA),
showing the charge distribution in the same colour scheme. The length of one turn of the helix and the
minor groove are shown with yellow dotted lines. (b) shows the structure of AbbA in cartoon
format with the side chain oxygens of glutamic acid residues 16, 29. 33 and 67
as red spheres and (d) is the same structure with the backbone of the DNA from
PDB 1BNA superimposed in yellow.
Figure taken
from Tucker, A.T. et al (2014) .
Few DNA mimics
have been discovered but they share the tactic of using negatively charged
residues (glutamic acids and aspartic acids) to present similar bonding
opportunities to the DNA backbone phosphates. It would seem that, faced with
the challenge of competing with the strongly charged backbone of DNA, the
mimics are following the logic that if you can't beat 'em, join 'em.
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