Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, United Kingdom.
Protein Sci. 2013 May;22(5):628-40. doi: 10.1002/pro.2247. Epub 2013 Mar 26.
YgjD from COG0533 is amongst a small group of highly conserved proteins present in all three domains of life. Various roles and biochemical functions (including sialoprotease and endonuclease activities) have been ascribed to YgjD and orthologs, the most recent, however, is involvement in the post transcriptional modification of certain tRNAs by formation of N6-threonyl-adenosine (t⁶A) at position 37. In bacteria, YgjD is essential and along with YeaZ, YjeE, and YrdC has been shown to be 'necessary and sufficient' for the tRNA modification. To further define interactions and possible roles for some of this set of proteins we have undertaken structural and biochemical studies. We show that formation of the previously reported heterodimer of YgjD-YeaZ involves ordering of the C-terminal region of YeaZ which extends along the surface of YgjD in the crystal structure. ATPγS or AMP is observed in YgjD while no nucleotide is bound on YeaZ. ITC experiments reveal previously unreported binary and ternary complexes which can be nucleotide dependent. The stoichiometry of the YeaZ-YgjD complex is 1:1 with a K(D) of 0.3 µM. YgjD and YjeE interact only in the presence of ATP, while YjeE binds to YgjD-YeaZ in the presence of ATP or ADP with a K(D) of 6 µM. YgjD doesn't bind the precursors of t⁶A, threonine, and bicarbonate. These results show a more complex set of interactions than previously thought, which may have a regulatory role. The understanding gained should help in deriving inhibitors of these essential proteins that might have potential as antibacterial drugs.
YgjD 是 COG0533 中的一种高度保守蛋白，存在于所有三个生命领域中。已经赋予了 YgjD 和同源物各种作用和生化功能（包括唾液酸酶和内切核酸酶活性），然而，最近的研究表明，YgjD 参与了某些 tRNA 的转录后修饰，通过在第 37 位形成 N6-苏氨酸-腺苷（t⁶A）。在细菌中，YgjD 是必需的，并且与 YeaZ、YjeE 和 YrdC 一起被证明是 tRNA 修饰的“必需和充分”。为了进一步定义这组蛋白质的一些相互作用和可能的作用，我们进行了结构和生化研究。我们表明，先前报道的 YgjD-YeaZ 异二聚体的形成涉及 YeaZ 的 C 末端区域的有序排列，该区域在晶体结构中沿 YgjD 的表面延伸。在 YgjD 中观察到 ATPγS 或 AMP，而 YeaZ 上没有结合核苷酸。ITC 实验揭示了以前未报道的二元和三元复合物，这些复合物可能依赖核苷酸。YeaZ-YgjD 复合物的化学计量比为 1:1，K(D)为 0.3 µM。只有在存在 ATP 的情况下，YgjD 和 YjeE 才相互作用，而 YjeE 在存在 ATP 或 ADP 的情况下与 YgjD-YeaZ 结合，K(D)为 6 µM。YgjD 不结合 t⁶A、苏氨酸和重碳酸盐的前体。这些结果表明，相互作用比以前想象的更为复杂，可能具有调节作用。获得的理解应该有助于开发这些必需蛋白的抑制剂，这些抑制剂可能具有作为抗菌药物的潜力。
