Interaction Of Antitoxin SO₁445 With Promoter DNA And Its Structural Basis

The Toxin-Antitoxin?TA?system is abundant in most of the chromosomes of bacteria and archea as well as phage genome.The TA system plays many specific roles,such as inducing cell dormancy in response to various stress responses,phage inhibition,gene regulation,and tolerance to antibiotics.The Type ? toxin-antitoxin system is best studied in five types due to its ubiquity in the genomes.The Type ? TA system functions in a way that both genes are translated into proteins with the antitoxin inhibiting the toxicity of the toxin through protein-protein interactions.Antitoxin proteins are composed of two domains:a C-terminal domain and an N-terminal domain.The antitoxin C-terminal domain binds toxin and neutralizes toxin toxicity.The N-terminal domain binds to the promoter DNA and participates in the negative feedback regulation of TA gene transcription.Currently,details of antitoxin participating negative feedback regulation are scarce.Therefore it is necessary to study the structures of antitoxins and reveal their structure-function relationships so as to clarify their action mechanisms.SO₁444/SO-1445 toxinantitoxin system is a pair of type ? toxinantitoxin system discovered by Xiaoxue Wang group of the South China Sea Institure of Oceanology,Chinese Academy of Science.Their results show that antitoxin SO₁445 binds to a specific palindrome sequence in its own promoter,and in some stains there is SO₁445only with the toxin absence.This study attempted to reveal its functional mechanism by investigating the interaction between the antitoxin SO₁445 and the promoter DNA and the structure of the complex.The N-terminal domain of SO₁445 binding to the promoter DNA?consisting of 58 residues,named SO₁445-N-58?was identified by sequence alignment and secondary structure prediction,and it was separately cloned,recombinantly expressed and the protein was purified.The interaction between full-length SO₁445 and its N-terminal structure?SO₁445-N-58?with the specific promoter DNA palindrome?19 bp dsDNA?was detected by ITC,the structure of SO₁445 and SO₁445-N-58 and the structure of the proteins complex with promoter were analyzed by NMR and X-ray crystallography.The results of ITC showed that there was a significant interaction between SO₁445 and 19 bp dsDNA and the binding ratio was 4:1,but the interaction between SO₁445-N-58 and the19 bp dsDNA was not significant.The NMR,DLS,CD and SEC results showed that the full-length protein and the N-terminal segment both interacted with the DNA.The NMR data showed that the structures of SO₁445 and SO₁445-N-58,as well as the structure of their complex with the 19 bp dsDNA,respectively,were not suitable for resolution by NMR.SO₁445-N-58 could grow crystals under certain conditions.SO₁445-N-58 can produce crystals under specific conditions.The crystal structure is very similar to that of other antitoxins.The crystallization conditions for the complexes of SO₁445-N-58 and SO₁445 with the 19 bp dsDNA are still in progress.Our results indicate that the structure of SO-1445's C-terminal segment is relatively disordered,which promotes the binding of the N-terminal segment to DNA,and the binding induces the C-terminal segment folding from disordered to helical structure.These properties help us to better understand the synergy between the antitoxin N-terminal and C-terminal segments in their function,the details of which are to be clarified.