DNA binding reveals hidden interdomain allostery of a MazE antitoxin from Mycobacterium tuberculosis.

Type II toxin-antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA-DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.

The structural and functional investigation of the VapBC43 complex from Mycobacterium tuberculosis.

Toxin - Antitoxin systems are crucial for bacterial survival against harsh circumstances such as antibiotic treatment. The VapBC systems are the most abundant Toxin-Antitoxin systems among the Toxin - Antitoxin systems in the Mycobacterium tuberculosis. The VapBC43 system is one of them, which is related to the response to the vancomycin treatment. However, the structure of the VapBC43 complex remained unknown. Here, we present the crystal structure of the VapBC43 complex in which a single VapB43 molecule binds to the VapC43 dimer. The electrophoretic mobility shift assay shows that the VapB43 can bind to its promoter DNA. In addition, this structure reveals that the VapC43 contains a PIN (PilT N-terminus) domain motif which is essential for ribonuclease activity but has less conserved acidic residues than other homologs. The results of ribonuclease assays show that the VapC43 exhibits ribonuclease activity despite the lack of acidic residues which are well conserved in a PIN domain superfamily. Based on the previous finding that the VapBC43 contributes to the survival of Mycobacterium tuberculosis under vancomycin treatment, the structural information of the VapBC43 complex may enable the development of the inhibitor of VapC43 that can be used as an adjuvant for vancomycin therapy against M. tuberculosis.

Structural and functional analysis of the D-alanyl carrier protein ligase DltA from Staphylococcus aureus Mu50.

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S. aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S. aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.

Crystal structure of the YoeBSa1-YefMSa1 complex from Staphylococcus aureus.

Toxin-antitoxin (TA) systems are ubiquitously found in bacteria and are related to cell maintenance and survival under environmental stresses such as heat shock, nutrient starvation, and antibiotic treatment. Here, we report for the first time the crystal structure of the Staphylococcus aureus TA complex YoeBSa1-YefMSa1 at a resolution of 1.7 Å. This structure reveals a heterotetramer with a 2:2 stoichiometry between YoeBSa1 and YefMSa1. The N-terminal regions of the YefMSa1 antitoxin form a homodimer characteristic of a hydrophobic core, and the C-terminal extended region of each YefMSa1 protomer makes contact with each YoeBSa1 monomer. The binding stoichiometry of YoeBSa1 and YefMSa1 is different from that of YoeB and YefM of E. coli (YoeBEc and YefMEc), which is the only structural homologue among YoeB-YefM families; however, the structures of individual YoeBSa1 and YefMSa1 subunits in the complex are highly similar to the corresponding structures in E. coli. In addition, docking simulation with a minimal RNA substrate provides structural insight into the guanosine specificity of YoeBSa1 for cleavage in the active site, which is distinct from the specificity of YoeBEc for adenosine rather than guanosine. Given the previous finding that YoeBSa1 exhibits fatal toxicity without inducing persister cells, the structure of the YoeBSa1-YefMSa1 complex will contribute to the design of a new category of anti-staphylococcal agents that disrupt the YoeBSa1-YefMSa1 complex and increase YoeBSa1 toxicity.

Two distinct mechanisms of transcriptional regulation by the redox sensor YodB.

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.