Phosphorylation-mediated conformational switch regulates human SLFN11

Human Schlafen 11 (SLFN11) serves as a predictive biomarker in cancer as it sensitizes cells to DNA damaging agents by irreversibly blocking stalled replication forks. SLFN11 further acts as an interferon-inducible antiviral restriction factor that targets translation in a codon-usage-dependent manner. However, the regulation of the SLFN11 functions and enzymatic activities remains enigmatic. Here, we present a structural and biochemical explanation for the regulation of SLFN11 by phosphorylation. A cryo-electron microscopy (cryo-EM) structure of the SLFN11 phosphomimetic mutant S753D revealed that it adopts a monomeric conformation, allowing ATP binding, yet loses its ability to bind single-stranded DNA (ssDNA). Cryo-EM structures of SLFN11 bound to tRNA-Leu and tRNA-Met give insights into tRNA binding and cleavage, as well as its regulation by phosphorylation at S219 and T230. Thus, the phosphorylation site S753 serves as a conformational switch, regulating SLFN11 dimerization, as well as ATP and ssDNA binding, while S219 and T230 regulate tRNA recognition and nuclease activity.