Osinski, A.; Mayro, B.; Lopez, V. A.; Schrad, J. R.; Choi, H.; Tomchick, D. R.; Pawlowski, K.; Forsberg, K. J.; Shahmoradian, S.; Tagliabracci, V. S.

Across all domains of life, organisms exploit NAD+ metabolism as a central line of defense against invading pathogens. Here, we show that domain of unknown function 4062 (DUF4062) is a widespread family of TIR-like NADases that hydrolyze NAD+ to ADP-ribose and nicotinamide. In bacteria, DUF4062 homologs form a previously unrecognized antiphage defense system, which we name Swaro[z]yc, that assembles with the phage portal into a supramolecular NADase complex to induce abortive infection. In eukaryotes, DUF4062 is found in TEP1, which we demonstrate functions as an active NADase within the RNA vault, an enigmatic organelle-like structure. Single-particle cryo-electron microscopy reveals ADP-ribose bound within the shoulder of both reconstituted and human brain vaults, while cryo-electron tomography positions TEP1 along the central axis at the shoulder. Thus, TEP1, like bacterial Swaro[z]yc, functions by depleting NAD+, providing new insight into the long-standing mystery of vault function.