Structure of Ribosome-Inactivating Protein from Mirabilis jalapa and Its L12-Stalk-Dependent Inhibition of Escherichia coli Ribosome

<i>Mirabilis</i> antiviral protein (MAP) is the type I ribosome-inactivating protein (RIP), which consists of an RNA <i>N</i>-glycosylase domain with no carbohydrate-binding domain. Unlike many RIPs, such as ricin or trichosanthin, which inactivate eukaryotic ribosomes, MAP also inactivates the <i>E. coli</i> ribosome by cleaving the <i>N</i>-glycosidic bond at A2660 of 23S ribosomal RNA. The structure of the wild-type MAP has not been revealed yet. Here, we expressed, purified, and crystallized the plural recombinant MAPs, including both E168Q and R171Q mutations (MAP-EQRQ) in <i>E. coli</i>, and determined the crystal structure of MAP-EQRQ at 2.1 Å resolution. According to the predicted structure with RNA (sarcin-ricin loop) and the mutant protein's activities using quantitative RT-PCR, we showed that residue R171 at the active site of MAP is a key residue to form the stable complex with target adenine. Furthermore, we showed that MAP bound the C-terminal domains of eukaryotic P2-stalk as well as <i>E. coli</i> L12-stalk.