Structural and functional mechanisms by which bacteria activate SspE through DNA vulcanization modification to resist phage infestation

Recently, the team of Professor Wu Geng from the School of Life Science and Technology/State Key Laboratory of Microbial Metabolism of Shanghai Jiao Tong University, together with the team of Professor Wang Lianrong and Professor Chen Shi of Wuhan University, elucidated the structural and functional mechanism of bacteria such as Streptomyces and other bacteria activating the nuclease activity of SspE protein in bacteria through phosphorsulphylation modification on DNA, and eliminating the phage DNA that invaded bacteria, thereby resisting phage infection
.
This study clarifies how bacteria activate SspE protein through DNA phosphosulfylation modification to achieve resistance to phage infection, and deepens people's understanding
of the function of DNA phosphosulfylation modification 。 The study was published in Nature Communications under the title "A coupled recognition-activating nicking mechanism underlying the DNA phosphorothioate-sensing antiphage defense barrier by SspE
.
" Wu Geng, Wang Lianrong and Professor Chen Shi are co-corresponding authors
.
This article is the structural mechanism of bacterial recognition DNA vulcanization modification published by Wu Geng's team in Nature Communications in 2018, the structure of SspB and SspE of type II DNA vulcanization modification published in Nature Microbiology in 2020, and the structure of SspA of type II DNA vulcanization modification published in mBio in 2020.
Continuation and expansion
of DndE structures modified by type I DNA vulcanization published in mBio 2022.

DNA phospheusulfuration modifications include two types: double-stranded type I modifications mediated by proteins encoded by the dndABCDE gene cluster and single-stranded type II modifications
mediated by proteins encoded by the sspABCD gene cluster.
SspE is a nuclease encoded by the sspE gene near the sspABCD gene cluster, 700-800 amino acids in length, consisting of
the N-terminal domain (NTD) with GTPase activity and the C-terminal domain (CTD) with inscribed nuclease activity.

Figure: Bacterial vulcanization modification of genomic DNA activates nuclease activity of SspE and cleaves phage DNA to resist phage infestation

First, by analyzing the 2.
7 Angstrom resolution crystal structure of the CTD domain of Streptomyces SspE and the 3.
4 Angstrom resolution crystal structure of the full-length SspE protein, this study found that the NTD and CTD domains of SspE form independently folded tertiary structures, respectively, and the NTD and CTD are connected by a short loop, while there is no tight bond
between NTD and CTD.
The NTD of SspE contains DGQQR motif, which identifies and hydrolyzes GTP
.
The CTD of Ssp contains HNH motif, which can be missing covalently closed circular DNA into open circular DNA, and mutating HNH motif on SspE-CTD will make it lose its resistance
to phage infection.

Then, by molecular docking calculation and GTPase enzyme activity experiments with site-directed mutants, it was found that SspE recognized bacterial phosphsulfurylation modified DNA through a more hydrophobic binding pocket on the surface of the NTD domain, and mutation of amino acid residues on this sulfide DNA-binding pocket would destroy the GTPase enzyme activity of SspE and its resistance to phage infection
.

Next, through non-denaturing gel electrophoretic migration experiments, this study found that the CTD domain of SspE can bind to phage DNA, and mutation of an important residue on a conserved, positively charged recessed surface on SspE-CTD will destroy the binding of SspE to phage DNA and cause SspE to lose its defense against
phage infection.
Moreover, the binding of SspE's CTD to DNA depends on the hydrolysis
of its NTD to GTP.

Finally, it is interesting to note that the study found that the R100E mutation caused SspE to lose GTPase enzyme activity and DNA dysenterase activity, but the R100 site was not at
the GTPase enzyme active site or the DNA dysenterase activity site 。 Therefore, this study analyzed the 3.
48 angstrom resolution crystal structure of the SspE-R100E mutant, and found that compared with the wild-type SspE, the CTD domain of the SspE-R100E mutant moved 2.
6-3.
9 angstroms towards the NTD domain as a whole, indicating that SspE has conformational changes
caused by the relative motion between the two domains.
Fluorescence resonance energy transfer experiments confirmed that the binding of bacterial phosphorsulphylated modified DNA and the hydrolysis of GTP did cause conformational changes
in the SspE protein in solution.

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