Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA

TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m7G) in tRNA. In tRNAs m7G 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 B. subtilis 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 Kd of TrmB and tRNAPhe 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-tRNAPhe complex structure.

TrmB属于I类S-腺苷甲硫氨酸（S-adenosylmethionine, SAM）依赖型甲基转移酶（methyltransferases, MTases）家族，可对tRNA中第7位鸟嘌呤残基进行甲基化修饰，生成m7G。在tRNA中，m7G最常分布于可变环的第46位，并与C13、U22形成三级碱基配对，同时在G46位点引入正电荷。TrmB/Trm8酶家族具有结构多样性，TrmB蛋白可分别以单体、同源二聚体及异源二聚体形式存在。截至目前，TrmB的确切酶促机制以及tRNA-TrmB复合物的晶体结构仍未阐明。本研究首次解析了枯草芽孢杆菌（B. subtilis）TrmB蛋白分别与SAM、SAH结合的复合物晶体结构。我们通过X射线晶体学（X-ray crystallography）分别解析了TrmB空载态、结合SAM及结合SAH的晶体结构，分辨率依次为1.9 Å（空载态）、2.5 Å（结合SAM）及3.1 Å（结合SAH）。晶体结构分析显示，Tyr193残基在SAM结合及甲基转移酶活性调控中发挥关键作用。通过荧光偏振实验，我们测得TrmB与tRNAPhe的解离常数Kd为0.12 µM ± 0.002 µM。基于发光法的甲基转移酶活性测定结果显示，在生理SAM浓度下，TrmB催化过程存在半位点反应性的协同效应。结合小角X射线散射（small-angle x-ray scattering, SAXS）、交联复合物质谱分析及分子对接实验获得的结构数据，我们最终解析了TrmB与tRNAPhe的复合物结构。