A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase
A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase
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DOI：
10.1073/pnas.1522891113
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发表时间：
2016-01-26
影响因子：
11.1
通讯作者：
Haeggstrom, Jesper Z.
中科院分区：
文献类型：
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作者：
Brock, Joseph S.;Hamberg, Mats;Haeggstrom, Jesper Z.
微粒体前列腺素E-2合成酶1型（mPGES-1）负责促炎条件下强效脂质介质前列腺素E-2的形成，该酶作为药物靶点受到了相当大的关注。最近，一种高分辨率的人类mPGES-1晶体结构被提出，Ser-127被认为是氢键供体，稳定在辅助因子谷胱甘肽（GSH）内的硫代阴离子形成。我们将定点诱变和活性分析与结构动力学分析相结合，以探索这些假定的催化残基的功能作用。我们发现Ser-127不是活性所必需的，而Arg-126和Asp-49之间的相互作用对于催化是必不可少的。我们假设，这两种残基，除了结晶水，在酶的机制中起关键作用。在表征了大小或电荷保守突变Arg-126- gln， Asp-49- asn和Arg-126- lys后，我们推断在GSH硫酸盐形成过程中，晶体水作为一般碱，通过与Arg-126的相互作用来稳定，而Arg-126本身则由其与Asp-49的相互作用来调节。我们随后在晶体结构中发现了隐藏的构象集合，这些构象集合与我们的生化数据密切相关。由此产生的接触信号网络将Asp-49连接到参与GSH结合的远端残基，并且依赖于配体。我们的工作对开发高效的mPGES-1抑制剂，潜在的抗炎和抗癌药物具有广泛的意义。
Microsomal prostaglandin E-2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E-2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.