Department of Molecular Structural Biology, Institute of Microbiology and Genetics, GZMB, Georg August University Göttingen, Göttingen, Germany.
Institute for X-Ray Physics, Georg August University Göttingen, Göttingen, Germany.
RNA Biol. 2021 Dec;18(12):2466-2479. doi: 10.1080/15476286.2021.1925477. Epub 2021 May 19.
TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (mG) in tRNA. In tRNAs mG is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant K of TrmB and tRNA was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNA complex structure.
TrmB 属于 I 类 S-腺苷甲硫氨酸（SAM）依赖性甲基转移酶（MTases），并在 tRNA 中引入一个甲基到位置 7（mG）的鸟嘌呤。在 tRNA 中，mG 最常见于可变环中的位置 46 处，并与 C13 和 U22 形成一个三级碱基对，在 G46 处引入一个正电荷。TrmB/Trm8 酶家族结构多样，因为 TrmB 蛋白存在单体、同源二聚体和异源二聚体形式。到目前为止，确切的酶促机制以及 tRNA-TrmB 晶体结构尚不清楚。在这里，我们首次展示了 TrmB 与 SAM 和 SAH 复合物的晶体结构。通过 X 射线晶体学确定了 TrmB apo 和与 SAM 和 SAH 复合物的晶体结构，分辨率分别为 1.9 Å（apo）、2.5 Å（SAM）和 3.1 Å（SAH）。获得的晶体结构表明 Tyr193 在 SAM 结合和 MTase 活性中很重要。通过荧光偏振，确定了 TrmB 和 tRNA 的解离常数 K 为 0.12 µM ± 0.002 µM。基于发光的甲基转移酶活性测定显示，在生理 SAM 浓度下，TrmB 催化过程中存在协同作用，半位反应性。从小角 X 射线散射（SAXS）、交联复合物的质谱和分子对接实验中检索到的结构数据，确定了 TrmB-tRNA 复合物的结构。
