SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association–dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

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Cyclic ADP ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via 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 leading to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan essential for cyclization. We show that 3′cADPR is an activator of ThsA effector proteins from bacterial anti-phage defense systems 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.

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MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.

The innate immune system consists of pattern recognition receptors (PRRs) that detect pathogenand endogenous danger-associated molecular patterns (PAMPs and DAMPs), initiating signaling pathways that lead to the induction of cytokine expression, processing of pro-inflammatory cytokines, and induction of cell-death responses. An emerging concept in these pathways and associated processes is signaling by cooperative assembly formation (SCAF), which involves formation of higher order oligomeric complexes, and enables rapid and strongly amplified signaling responses to minute amounts of stimulus. Many of these signalosomes assemble through homotypic interactions of members of the death-fold (DF) superfamily, Toll/IL-1 receptor (TIR) domains, or the RIP homotypic interaction motifs (RHIM). We review the current understanding of the structure and function of these domains and their molecular interactions with a particular focus on higher order assemblies.

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.

Background: The regulation of ACOT12 by ADP and ATP is thought to occur through oligomerization. Results: The structures of apo-ACOT12 and ADP-bound ACOT12 reveal new insights into regulation. Conclusion: ACOT12 is a homotrimer and neither ADP nor ATP alter the oligomeric state of the protein.Significance: These findings provide the first structural insights in the regulation of this enzyme family.