Varshney Dhaval, Petit Alain-Pierre, Bueren-Calabuig Juan A, Jansen Chimed, Fletcher Dan A, Peggie Mark, Weidlich Simone, Scullion Paul, Pisliakov Andrei V, Cowling Victoria H
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Nucleic Acids Res. 2016 Dec 1;44(21):10423-10436. doi: 10.1093/nar/gkw637. Epub 2016 Jul 15.
Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.
真核生物中mRNA的成熟和翻译需要添加7-甲基鸟苷帽。在脊椎动物中，帽甲基转移酶，即RNA鸟嘌呤-7甲基转移酶（RNMT），有一个激活亚基，即RNMT激活小蛋白（RAM）。在此，我们报道了与RAM激活结构域结合的人源RNMT的首个晶体结构。相对无结构且带负电荷的RAM与RNMT上远离活性位点的带正电荷的表面凹槽结合。这导致了与RAM共同进化且RAM结合所必需的RNMT叶结构的稳定。结构导向的诱变和分子动力学模拟表明，RAM稳定了RNMT叶和相邻α-螺旋铰链的结构及定位，从而使与活性位点中的底物接触的A螺旋实现最佳定位。通过生物物理和生化方法，我们观察到RAM增加了甲基供体S-腺苷甲硫氨酸（AdoMet）向RNMT的募集。因此，我们报道了RAM变构激活RNMT的机制，使其能够作为mRNA帽甲基化的分子变阻器发挥作用。
