Structural Basis Of HepT-MntA Toxin-antitoxin System In Thermococcus Cleftensis CL1

The toxin-antitoxin（TA）system is a genetic element widely present in bacteria and archaea.This system usually consists of a toxin and an antitoxin that inhibits the toxin.Toxins inhibit bacterial growth through a variety of pathways,while antitoxins maintain the normal physiological activities of cells by inhibiting the toxic effects of toxins.According to the mode of action and nature of the antitoxin,the TA system can be divided into types I-VIII.Among them,the neutralization of toxin toxicity in the type VII TA system depends on its post-translational modification by the antitoxin.It has been found that the neutralization of the toxin HepT toxicity in the TA system of Shewanella oneidensis depends mainly on the polyadenylation modification of the antitoxin MntA,classifying HepT-MntA as a type VII TA system.While analyzing the sequence homology of HepT and MntA in the TA system of S.oneidensis,a similar TA system was found in Thermococcus cleftensis CL1.Although the mnt-hepn gene of this type of system is widely present in the sequenced microorganisms,the relevant biological functions and neutralization mechanisms have been relatively little studied.Therefore,this study was carried out to investigate the biological functions of the TA system of T.cleftensis CL1,and the details and results of the study are as follows:（1）Bioinformatics analysis revealed that the T.cleftensis CL1 genomic genes CL1＿0070 and CL1＿0071 encode proteins with the conserved motifs GSX₁₀DVD and RX₄HAY of the MNT structural domain and HEPN structural domain,respectively,and we hypothesize that CL1＿0070-CL1＿0071 form a type VII TA system named HepT_CL1-MntA_CL1.（2）In this experiment,the E.coli expression system was used for the expression of the target protein,and it was clear through bacterial toxicity identification experiments that the CL1＿0071 gene encodes the toxin HepT_CL1 and the CL1＿0070 gene encodes the antitoxin MntA_CL1,and the MntA_CL1 can neutralize the toxicity of the HepT_CL1,The experimental results show that HepT_CL1-MntA_CL1 is a TA system.（3）Co-expression of（WT）MntA_CL1/HepT_CL1 protein with significantly higher nucleic acid values compared to the A260/A280 values of the toxin and antitoxin monoproteins observed by gel exclusion chromatography purification process,implying that the toxin HepT_CL1 protein may be modified by adenosylation.We identified the molecular weight of toxin HepT_CL1 protein in co-expressed（WT）MntA_CL1/HepT_CL1 protein by high perfor-mance liquid chromatography-mass spectrometry,and the value of increase in molecular weight of toxin HepT_CL1 Y82 residue corresponds to the theoretical value of AMP,and the data suggest that antitoxin MntA_CL1 adenylation modifies toxin HepT_CL1 Y82 site.In addition,the Y82 site（Y82F）of the toxin in the mutant HepT_CL1/MntA_CL1 complex was found to exhibit inhibition of E.coli growth by toxicity identification assays,suggesting that the Y82 site of the mutant toxin HepT_CL1 prevents adenylation modification and that adenylation of the toxin leads to its loss of toxicity.（4）In order to further explore the molecular mechanism of the loss of toxicity after adenylation of the toxin in the T.cleftensis CL1 TA system,We resolved the crystal structures of MntA_CL1,HepT_CL1 and co-expressed（WT）MntA_CL1/HepT_CL1 proteins by crystallographic methods.The structure of MntA_CL1 has the typical multimer folding mode of MNT,and the whole structure of HepT_CL1 is composed of two HEPNs to form a symmetrical V-shaped dimer.The crystal structure of HepT_CL1/MntA_CL1 binary complex presents a heterotetramer with a molecular symmetry unit of（HEPN）₂:（MNT）₂.By analyzing and mutating the key amino acids at the interface between MNT and HEPN in the binary complex structure,it was shown that four amino acids at the sites R69,R73,R88,and D97 play major functions in the recognition of MNT and HEPN.We also resolved the structure of mutant Y82F,which could not be modified by adenylation,and the mutant Y82F structure still showed the（HEPN）₂:（MNT）₂ tetrameric conformation.Comparison of the conformational changes of WT and mutant Y82F showed that the adenylation acidification of Y82 caused the Y82 loop of toxin HepT_CL1 to be pulled away from the RNase catalytic center,resulting in loss of toxin RNase activity and thus reduced toxicity.Taken together,both functional and structural biology studies suggest that the loss of protein toxicity of the toxin HepT_CL1 is due to the adenylation modification of its Y82 site by the antitoxin MntA_CL1.We finally elucidated the mechanism of action of antitoxin MntA as a nucleotidyl transferase in the T.cleftensis CL1 TA system using substrate ATP for adenylation modification of toxin HepT and identified HepT_CL1-MntA_CL1 as a novel type VII TA system.