Protonation states of the key active site residues and structural dynamics of the glmS riboswitch as revealed by molecular dynamics.
Protonation states of the key active site residues and structural dynamics of the glmS riboswitch as revealed by molecular dynamics.
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分子动力学揭示了关键活性位点残基的质子化状态和 glmS 核糖开关的结构动力学。
DOI：
10.1021/jp9109699
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发表时间：
2010-07-08
影响因子：
3.3
通讯作者：
Otyepka, Michal
中科院分区：
文献类型：
--
作者：
Banas, Pavel;Walter, Nils G.;Sponer, Jiri;Otyepka, Michal
GLMS催化核糖开关是许多革兰氏阳性菌中编码氨基葡萄糖-6-磷酸(GlcN6P)合成酶(GLMS)的mRNAs 5‘非翻译区的一部分。辅因子GlcN6P的结合诱导RNA的位点特异性自我切割。然而，GLMS反应形式的详细反应机理和质子化状态仍然不清楚。为了探索关键活性中心残基的主要质子化状态，我们进行了显式溶剂分子动力学模拟，涉及在可用晶体结构中观察到的三个关键活性中心部分的质子化状态：(I)鸟嘌呤G40(遵循T.tengcongensis编号)，(Ii)GlcN6P氨基/氨基，和(Iii)GlcN6P磷酸部分。我们发现去质子化的G40−似乎与观察到的GLMS活性中心结构不相容。我们的数据表明，G40的规范形式通过稳定裂解位点A-1(2‘-OH)亲核试剂的在线攻击构象而发挥结构作用，而不是更直接的化学作用。此外，我们观察到GlcN6P磷酸部分质子化时辅助因子结合减弱，这解释了实验观察到的Km随pH降低而增加的现象。最后，我们讨论了辅因子结合及其与G65和G1嘌呤的相互作用在A-1(2‘-OH)顺排攻击构象结构稳定中的可能作用。根据确定的反应前驱体的主要质子化状态，我们提出了一个自裂解机理的假说，其中A-1(2‘-OH)被剪式磷酸的G1(Pro-RP)非桥氧激活为亲核试剂，而GlcN6P的氨基则作为总的酸质子化G1(O5’)离开基团。
The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous Gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, detailed reaction mechanism as well as protonation state of glmS reactive form remains still elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the T. tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40− seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase of Km with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. Based on the identified dominant protonation state of the reaction precursor, we propose a hypothesis of self-cleavage mechanism, in which A-1(2'-OH) is activated as nucleophile by the G1(pro-Rp) non-bridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group.
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影响因子：
4.8
作者：
Banas, Pavel;Jurecka, Petr;Walter, Nils G.;Sponer, Jiri;Otyepka, Michal
通讯作者：
Otyepka, Michal
影响因子：
2.9
作者：
Almlof, Martin;Ander, Martin;Aqvist, Johan
通讯作者：
Aqvist, Johan
影响因子：
4.5
作者：
Breaker, RR;Emilsson, GM;Sudarsan, N
通讯作者：
Sudarsan, N
影响因子：
64.8
作者：
Batey, RT;Gilbert, SD;Montange, RK
通讯作者：
Montange, RK
影响因子：
4.5
作者：
Ditzler, Mark A.;Sponer, Jiri;Walter, Nils G.
通讯作者：
Walter, Nils G.