Fig. 2: The TIR assembly is essential for NADase activity.

a The tetramerization of TIR domains. The four TIR-APAZ/Ago units (A/A’/B/B’) are colored in purple, orange, slate, and wheat, respectively. The lower panel shows the intrastrand (green box) and interstrand (black box) contacts formed by TIR domains. The flipped-out BB-loops (purple and orange) may indicate an active state. b Detailed insights into the intrastrand interactions. Key interacting residues are shown in stick representation. c Detailed insights into the interstrand interactions. Key interacting residues are shown in stick representation. d Native PAGE of the wild-type (WT) and mutant TIR-APAZ/Ago complexes. The TIR-APAZ/Ago complex could form a higher-order assembly in the presence of target ssDNA compared to the TIR-APAZ/Ago–gRNA RNP complex. Key residues in the TIR assembly interface were mutated. e NAD⁺ hydrolysis by WT or mutant TIR-APAZ/Ago complexes. Mutations of the residues in the TIR assembly interface decreased NAD⁺ cleavage. The E77A catalytic mutant was used as a control. ɛ-NAD⁺ was used as the substrate. Fluorescence intensity (410 nm/310 nm) was measured. The columns are colored the same as the corresponding residues in b and c. All assays were performed in triplicate, and error bars represent the standard deviations. f Superposition of TIR domains in the four units of target DNA-bound TIR-APAZ/Ago complex (the same color scheme as in a is used) and in the TIR-APAZ/Ago–gRNA RNP complex (white). g Structural comparison of the TIR domains (purple and slate) in TIR-APAZ/Ago–gRNA–ssDNA quaternary complex and the 1AD-bound TIR domains (white and gray) in human SARM1 (PDB ID: 7NAK). 1AD (cyan), the inhibitor of SARM1, has a chemical structure similar to NAD⁺. Residues in the substrate-binding pocket are shown in stick representation. h In vitro NAD⁺ degradation assays of WT or mutant TIR-APAZ/Ago protein complexes. Mutations of the residues in the putative substrate-binding pocket substantially weakened or abolished NAD⁺ consumption. The columns are colored the same as the corresponding residues in g. All assays were performed in triplicate, and error bars represent the standard deviations.