Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π^∗ interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

来自超嗜热菌（存在于沸水条件下的有机体）的蛋白质的稳定性通过降低构象灵活性而实现，这是通过各种机制实现的。由天冬氨酰/天冬酰胺残基的侧链与后续残基的骨架酰胺-NH 的翻译后环化产生的琥珀酰亚胺 (SNN) 将抑制扭转角 Ψ，并可作为超热稳定性的新途径。然而，这种琥珀酰亚胺通常易于水解，转化为天冬氨酰或β-异天冬氨酰残基。在这里，我们介绍了Methanocaldococcus jannaschii的晶体结构谷氨酰胺氨基转移酶，并使用增强的采样分子动力学模拟，解决了其增加的热稳定性的机制，高达 100°C，这是由位置 109 处意外稳定的琥珀酰亚胺残基赋予的。SNN109 对水解的稳定性被认为是由其静电屏蔽引起的通过其后续残基 Asp110 的侧链羧酸酯基团，以及通过SNN109 与其前面的残基 Glu108 之间的n → π ^{*相互作用，这两者都阻止了水进入 SNN。}稳定的琥珀酰亚胺残基诱导α的形成- 转角结构涉及 13 个原子的氢键，它锁定了局部构象，降低了蛋白质的灵活性。用 Φ 限制的脯氨酰残基替换 SNN 后蛋白质的去稳定化突出了琥珀酰亚胺残基在赋予酶超热稳定性方面的特异性。几种古细菌 GATase 中形成琥珀酰亚胺的三肽序列 (E(N/D)(E/D)) 的保守性强烈表明这种原本有害的翻译后修饰被改编为热稳定性的预兆。