Eduardo Tassoni Tsuchida (@eduardotassoni.bsky.social)

Phage terminase recognition by the bacterial immune sensors Avs2 and Upx Prokaryotes employ diverse defense strategies to detect and halt the progression of phage infection. Multiple defense systems sense phage proteins through direct binding, including antiviral STAND NTPases (Avs), which oligomerize upon target recognition to induce programmed cell death. The widespread Avs2 family was previously shown to detect the large terminase subunit of tailed phages, but the mechanism of terminase sensing was unknown. Here, we determine the structural basis of terminase recognition by Avs2 from Escherichia coli (EcAvs2). A cryo-EM structure at 2.3 Å resolution reveals that EcAvs2 forms a flat, C4-symmetric tetramer in which each protomer is bound to a single terminase monomer. Terminase recognition is mediated by a large, shape complementary binding pocket in the EcAvs2 sensor domain, including specific contacts with an unexpected ATP molecule at the interface of EcAvs2 and terminase. Furthermore, we demonstrate that the defense protein Upx also recognizes diverse phage terminases, despite lacking sequence and structural homology to Avs. AlphaFold 3 models indicate that Upx binds an unfolded state of the core terminase ATPase domain, mediated by β-augmentation. These findings highlight the distinct modes of terminase recognition across structurally diverse defense proteins. ### Competing Interest Statement F.Z. is a scientific advisor and cofounder of Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Aera Therapeutics, and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant. The remaining authors have no competing interests to declare. National Institute of General Medical Sciences, 5T32GM007276, 5T32GM141828 Stanford Bio-X Helen Hay Whitney Foundation, https://ror.org/037ebw447 Howard Hughes Medical Institute, https://ror.org/006w34k90 Yang Tan Collective K. Lisa Yang and Hock E. Tan Molecular Therapeutics Center Broad Institute Programmable Therapeutics Donors BT Charitable Foundation G. Harold & Leila Y. Mathers Foundation, MF-2303-04116 Stanford University School of Medicine, https://ror.org/011pcwc98 https://www.biorxiv.org/content/10.64898/2026.04.30.721860v1.full.pdf+html