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.