Bisubstrate analogues as structural tools to investigate m6A methyltransferase active sites
Bisubstrate analogues as structural tools to investigate m6A methyltransferase active sites
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DOI：
10.1080/15476286.2019.1589360
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发表时间：
2019-06-03
期刊：
影响因子：
4.1
通讯作者：
Tisne, Carine
中科院分区：
文献类型：
--
作者：
Oerum, Stephanie;Catala, Marjorie;Tisne, Carine
RNA甲基转移酶（MTases）使用最常见的S-腺苷-L-甲硫氨酸（SAM）作为辅因子催化甲基转移到其RNA底物。由于RNA：蛋白质复合物结晶的困难，目前只有很少的RNA结合的MTase结构可用。缺乏复杂的结构导致对RNA识别模式和甲基化反应机制知之甚少。相反，许多辅因子结合的MTase结构是可用的，导致充分理解的蛋白质：辅因子识别，这可以指导模拟底物和辅因子结合的状态的双底物类似物的设计。这种双底物类似物是最近合成的蛋白质单甲基化腺嘌呤（m（6）A）的N6-原子。这些蛋白质包括大肠杆菌中的RlmJ。大肠杆菌和人METLL 3：胃14和胃L16的表达。作为一个概念验证，我们在这里测试的双底物类似物的能力，以模仿底物：辅因子结合状态在催化过程中，通过研究他们的结合RlmJ使用差示扫描荧光法，等温滴定量热法和X-射线晶体学。我们发现甲基化的腺嘌呤碱基结合在正确的口袋中，因此这些类似物可能被广泛用于研究m（6）A MTases的RNA识别和催化机制。两种双底物类似物以微摩尔亲和力结合RlmJ，并且可以作为针对m（6）A RNA MTases的抑制剂设计的起始支架。相同的类似物引起m（1）A RNA MTase，TrmK的解链温度的变化，表明非选择性蛋白质：化合物复合物的形成。因此，优化这些分子支架的m（6）A RNA MTase抑制应旨在增加选择性，以及亲和力。
RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m(6)A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m(6)A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m(6)A RNA MTases. The same analogues cause changes in the melting temperature of the m(1)A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m(6)A RNA MTase inhibition should aim to increase selectivity, as well as affinity.