Cryo-EM structures of autoinhibited and RNA-activated ZNFX1 helicase

Abstract

ZNFX1 is an RNA helicase that plays a critical role in host defense against viral infections. Recent studies further revealed that ZNFX1 harbors an unexpected RNA-ubiquitinating E3 ligase activity, raising fundamental questions about how RNA-binding activates this multifunctional enzyme. Here, we combine cryo-electron microscopy with biochemical analyses to define the structural basis of ZNFX1 regulation. In the absence of RNA, ZNFX1 adopts an autoinhibited conformation in which a blocking helix occludes the RNA-binding groove. It exists predominantly as autoinhibited monomers, and, at higher concentrations, assembles into short helical filaments composed of repeating tetramers. Binding of single-stranded RNA (ssRNA) remodels these higher-order assemblies into RNA-bound ZNFX1 monomers and dimers. The cryo-EM structure of the ssRNA-bound ZNFX1 dimer reveals that RNA binding displaces the blocking helix and induces large-scale conformational rearrangements that remodel the helicase core into an active configuration. Biochemical analyses validate the proposed RNA-binding interfaces and demonstrate ATP-dependent RNA unwinding by ZNFX1. Together, these findings support a regulatory mechanism in which autoinhibited ZNFX1 exists as monomers or concentration-dependent higher-order oligomers, whereas RNA binding promotes structural remodeling and helicase activation.