biostudies-literatureUnknown15(1)Gao XSETD3 is an essential host factor for the replication of a variety of enteroviruses that specifically interacts with viral protease 2A. However, the interaction between SETD3 and the 2A protease has not been fully characterized. Here, we use X-ray crystallography and cryo-electron microscopy to determine the structures of SETD3 complexed with the 2A protease of EV71 to 3.5 Å and 3.1 Å resolution, respectively. We find that the 2A protease occupies the V-shaped central cleft of SETD3 through two discrete sites. The relative positions of the two proteins vary in the crystal and cryo-EM structures, showing dynamic binding. A biolayer interferometry assay shows that the EV71 2A protease outcompetes actin for SETD3 binding. We identify key 2A residues involved in SETD3 binding and demonstrate tNature communications4176https://www.ebi.ac.uk/biostudies/studies/S-EPMC11099015biostudies-literatureThe EV71 2A protease occupies the central cleft of SETD3 and disrupts SETD3-actin interaction.PMC11099015Ding WWang BZhu KShang KGao XCui SWang JQin BWang LfalseThe EV71 2A protease occupies the central cleft of SETD3 and disrupts SETD3-actin interaction.SETD3 is an essential host factor for the replication of a variety of enteroviruses that specifically interacts with viral protease 2A. However, the interaction between SETD3 and the 2A protease has not been fully characterized. Here, we use X-ray crystallography and cryo-electron microscopy to determine the structures of SETD3 complexed with the 2A protease of EV71 to 3.5 Å and 3.1 Å resolution, respectively. We find that the 2A protease occupies the V-shaped central cleft of SETD3 through two discrete sites. The relative positions of the two proteins vary in the crystal and cryo-EM structures, showing dynamic binding. A biolayer interferometry assay shows that the EV71 2A protease outcompetes actin for SETD3 binding. We identify key 2A residues involved in SETD3 binding and demonstrate t2024-01-01T00:00:00Z2024 May2026-06-02T06:54:52.633Z2026-04-15T03:15:19.851ZS-EPMC110990153875517610.1038/s41467-024-48504-w