Molecular Mechanism Of Conditional Cooperativity Of Type Ⅱ Toxin-antitoxin System YoeB-YefM From Staphylococcus Aureus

Toxin-antitoxin(TA)systems are gene pairs within an operon,which consist toxin to regulate the key cellular processes and an antitoxin that neutralizes the activity of the cognate toxin.The ratio of toxin and antitoxin in some type II TA systems could be dynamically adjusted to autoregulate its own transcription with the change in growth conditions termed as conditional cooperativity.YoeB-YefM,the widespread type Ⅱtoxin-antitoxin(TA)module,binds to its own promoter to autoregulate its transcription:repress or induce transcription under normal or stress conditions,respectively.It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio.S.aureus is a human pathogen that causes most nosocomial infections.Interestingly,S.aureus present two YoeB-YefM paralogs(SaiYoeB-Sa1YefM,Sa2YoeB-Sa2YefM)that can be found in the same strains simulataneously.The two YoeB-YefM paralogs do not cross talk each other.Here we explore the molecular mechanism of conditional cooperativity in those two YoeB-YefM paralogs by X-ray crystallography and corresponding biochemical experiments.In the first paralogue(Sa1YoeB-Sa1YefM),we find that Sa1YoeB-Sa1YefM complex exists as two distinct oligomeric assemblies:heterotetramer(Sa1YoeBSa1YefM2-Sa1YoeB)and heterohexamer(Sa1YoeB-Sa1YefM2-Sa1YefM2-Sa1YoeB)with low and high promoter DNA-binding affinities,respectively.Crystal structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that Sa1YefM C-terminal domain undergoes disorder to order transition upon Sa1YoeB binding,which allosterically affects the conformation of N-terminal DNA-binding domain.At TA ratio of 1:2,unsaturated binding of Sa1 YoeB to the C-terminal regions of Sa1YefM dimer forms an optimal heterohexamer for DNA binding,and two Sa1YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5’-TTGTACAN6AGTACAA-3’ palindromic sequence,resulting in transcriptional repression.In contrast,at TA ratio of 1:1,binding of two additional Sa1YoeB molecules onto the heterohexamer induces the completely ordered conformation of Sa1YefM and disassembles the heterohexamer into two heterotetramers,which are unable to bind the promoter DNA optimally due to steric clashes,hence derepresses TA operon transcription.To understand the molecular mechanism how Sa2YoeB-Sa2YefM regulate its own transcription and how each paralog function independently,we solve the crystal structures of heterohexamer(Sa2YoeB2-Sa2YefM4),heterotetramer(Sa2YoeB2Sa2YefM2)and heterohexamer-DNA ternary complex(Sa2YoeB2-Sa2YefM4-DNA).Our structural and biochemical data demonstrate that both YoeB-YefM paralogous copies adopt similar mechanism for transcriptional autoregulation.For instance,hydrogen bond network between the two heterotrimers is critical for the heterohexameric state formation and optimal DNA-binding affinity.In contrast,the steric clashes due to the simultaneous binding of two heterotetramers to the adjacent promoter DNA sites disrupt DNA-binding affinity.Molecular diversity in the two paralogs was investigated by comparative analysis of interaction profile of YefM and YoeB and recognition pattern of YefM and DNA.First,the conformation and acting force of interface between toxin and antitoxin are unique for Sa2YoeB-Sa2YefM.Second,Sa2YefM could recognize the flanking nucleotide sequences "G" and "C" by residues Thr7 and Tyrl4 while Sa1YefM could recognize the flanking nucleotide "T"by residues Tyr6 and Ser7.These results provide the structural insight into the molecular diversity and independent function of each paralogous YoeB-YefM copy.These will further open the way to analyze the detailed molecular evolution of structural insulation in other TA paralogs.Together,our work provides detail molecular insights into understanding YoeBYefM complex mediated transcriptional autoregulation,which will help target this type II TA system to overcome antibiotics resistance in Staphylococcus aureus and other pathogenic bacteria.