Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD+ degradation - PubMed (original) (raw)
Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD+ degradation
Donghyun Ka et al. Nat Commun. 2020.
Abstract
The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD+ cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD+ cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD+ degradation.
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1. ThsA cleaves NAD+ into Nam and ADPR.
a NAD+ is degraded by ThsA, but not by ThsB. Data are presented as mean ± SEM for three independent experiments. b LC chromatograms of the NAD+ cleavage products. MS analyses of each chromatographic peak are presented in Supplementary Fig. 2. c ThsA hydrolyzes the Nam–ribosyl bond of NAD+. Molecular weights of NAD+, Nam, and ADPR are indicated in parentheses. d Kinetic parameters of NAD+ cleavage catalyzed by ThsA. Data are presented as mean ± SEM for four independent experiments. Source data are provided as Source Data file.
Fig. 2. Crystal structure of ThsA.
a A two-domain architecture of ThsA consisting of an N-terminal sirtuin-like domain (blue) and a C-terminal SLOG-like domain (cyan). b Structure of the N-terminal sirtuin-like domain in ThsA. c Structural alignment of the Rossmann-like fold portion of the ThsA N-terminal domain (blue) with a sirtuin protein, Thermotoga maritima CobB (PDB ID: 2H2G; gray). d Structure of the C-terminal SLOG-like domain of ThsA. e Common features of the SLOG superfamily shared by the C-terminal domain of ThsA. ThsA residues conserved in the STALD family within the SLOG superfamily are shown in stick representation. Secondary structural elements of ThsA common to the SLOG superfamily are also indicated.
Fig. 3. NAD+ cleavage activity is intrinsic to the N-terminal sirtuin-like domain of ThsA.
a Structural alignment of the ThsA N-terminal domain (blue) with a sirtuin deacetylase, T. maritima CobB (PDB ID: 2H2F; gray) bound to a co-substrate, NAD+ (green). The essential zinc ion in the CobB structure is represented as a gray sphere. b Close-up view of the NAD+ binding site. Asn112 and His152 of ThsA align with sirtuin residues interacting with NAD+. c Sequence alignment of the N-terminal domain of ThsA with sirtuin proteins. Asn112 and His152 of ThsA are marked with asterisks. White character in red box indicates strict identity. Red character and blue frame represent similarity in a group and across groups, respectively. Alignment of the full-length amino acid sequences is presented in Supplementary Fig. 6. d Asn112 and His152 of the N-terminal sirtuin-like domain are critical for NAD+ hydrolysis of ThsA. N112A and H152A mutations of ThsA abolished NAD+ cleavage activity of ThsA. The graph for the wild-type ThsA (Fig. 1a) is shown again as a control for comparison. Data are normalized to the values measured at 0 min for each protein and presented as mean ± SEM for three independent experiments. Source data are provided as Source Data file.
Fig. 4. Crystal structure of ThsB.
a Overall structure of ThsB containing a TIR domain (red) and an additional β-sheet (orange). Structures of the TIR domain exhibiting the Rossmann-like fold (b) and the additional antiparallel β-sheet (c) of ThsB. d Structural alignment of the TIR domain of ThsB (red) with Hydra vulgaris toll-receptor-related 2 (PDB ID: 4W8G; gray).
References
- Ofir G, Sorek R. Contemporary phage biology: from classic models to new insights. Cell. 2018;172:1260–1270. -PubMed
- Weinbauer MG. Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 2004;28:127–181. -PubMed
- Fuhrman JA. Marine viruses and their biogeochemical and ecological effects. Nature. 1999;399:541–548. -PubMed
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