Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling

Cyclic adenosine diphosphate (ADP)–ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2′cADPR) and v2-cADPR (3′cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2′cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3′cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3′cADPR in bacteria as an antiviral and plant immunity–suppressing signaling molecule. Description Production of small signaling isomers Isomers of the small signaling molecule cyclic ADP ribose (cADPR) are produced when bacteria and plants respond to pathogens. Manik et al. found that cyclization is managed by Toll/interleukin-1 receptor (TIR) domain proteins that are themselves activated by conformational changes. One cyclic isomer, 3′cADPR, activates bacterial anti-hage defense systems in bacteria and suppresses immunity in plants. —PJH The production of signals in bacterial and plant immune responses reveals diversity in small signaling molecules. INTRODUCTION Organisms from bacteria to animals and plants must defend themselves against pathogens. Homologous protein motifs exist in immune pathways of all organisms. One such motif is the TIR domain, named after the mammalian immune receptors—Toll-like receptors and interleukin-1 receptors—where it was first identified. Two properties are shared among most TIR domains from all organisms: the ability to self-associate and enzymatic activity involving the cleavage of nicotinamide adenine dinucleotide (oxidized form) (NAD+). NAD+ is a metabolite with redox properties that has roles in many cellular processes. In some cases, cleavage of NAD+ leads to the production of cyclic adenosine monophosphate (ADP)–ribose (cADPR) isomers. RATIONALE In bacteria, NAD+-cleavage activity by TIR domain–containing proteins plays a role in defense signaling, as well as suppression of host immunity. One corresponding pathway is termed the Thoeris defense system. This signaling pathway protects bacteria against phage infection and involves the thsA and thsB genes. Upon phage infection, ThsB (a TIR-domain protein) cleaves NAD+ and produces a cADPR isomer, which activates ThsA-mediated killing of the infected cell, thus protecting the bacterial population. Another bacterial protein that produces a cADPR isomer is HopAM1, the TIR-domain effector protein from Pseudomonas syringae DC3000, which is involved in suppressing plant immunity. The chemical structures and mechanisms of action of the responsible cADPR isomers were unknown before this work. Our aim was to determine the chemical structures of cADPR isomers, the structural basis of their production by bacterial TIR domains, and their mechanism of action in Thoeris defense signaling and suppression of plant immunity. RESULTS Using a combination of methods, including nuclear magnetic resonance (NMR), mass spectrometry, and crystallography, we show that the cADPR isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of TIR domains that produce cADPR isomers, as determined by crystallography and cryo–electron microscopy, reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. Using crystallography and biophysical approaches, we show that one of the cADPR isomers (3′cADPR) is an activator of Thoeris ThsA proteins responsible for antiphage defense, by inducing a change in its tetrameric state. We also show that the same cADPR isomer is a suppressor of plant immunity when produced by the effector HopAM1. CONCLUSION Collectively, our results reveal the molecular basis of cADPR isomer production. The 2′cADPR and 3′cADPR differ only in the location of the O-glycosidic bond between the ribose moieties in ADPR. These compounds add to the growing list of signaling molecules identified in immune pathways that involve proteins containing TIR domains and may represent intermediates in their synthesis or signaling molecules with their own distinctive activities. Our results establish the 3′cADPR isomer produced by bacterial TIR domain–containing proteins as an antiviral and plant immunity–suppressing signaling molecule. Diverse immune roles of bacterial cADPR isomers. Bacteria have TIR domain–containing proteins that cleave NAD to produce cyclic ADPR isomers with different cyclic linkages. One of these molecules, 3′cADPR, has roles in diverse immunity pathways. It acts as an activator of the Thoeris antiphage defense system by binding to the protein ThsA. When produced by the effector HopAM1 from the plant pathogen P. syringae, it suppresses plant immunity.