Michalska Karolina, Gucinski Grant C, Garza-Sánchez Fernando, Johnson Parker M, Stols Lucy M, Eschenfeldt William H, Babnigg Gyorgy, Low David A, Goulding Celia W, Joachimiak Andrzej, Hayes Christopher S
Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Nucleic Acids Res. 2017 Sep 29;45(17):10306-10320. doi: 10.1093/nar/gkx700.
Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.
接触依赖性生长抑制（CDI）是一种细胞间竞争机制，革兰氏阴性菌通过V型分泌系统交换多态性毒素。在此，我们展示了来自大肠杆菌NC101的CDI毒素与其同源免疫蛋白和延伸因子Tu（EF-Tu）形成的三元复合物的结构。该毒素仅与EF-Tu的结构域2结合，部分重叠与氨酰-tRNA（aa-tRNA）3'-末端相互作用的位点。该毒素具有独特的核糖核酸酶活性，可从含有鸟嘌呤判别核苷酸的tRNA上切割单链3'-末端。在体外，需要EF-Tu来支持这种tRNA酶活性，这表明该毒素在GTP·EF-Tu·aa-tRNA复合物的背景下特异性切割底物。然而，将毒素结构域叠加到先前解析的GTP·EF-Tu·aa-tRNA结构上，发现与aa-tRNA和EF-Tu的开关I区域都存在潜在的空间冲突。此外，该毒素会诱导EF-Tu的构象变化，取代与aa-tRNA 3'-末端腺苷酸形成关键盐桥接触的β-发夹环。总之，这些观察结果表明，该毒素重塑GTP·EF-Tu·aa-tRNA复合物，以使aa-tRNA的3'-末端游离，从而进入核酸酶活性位点。
