Department of Biochemistry, 307 Research Dr., Box 3711, Duke University Medical Center, Durham, NC, 27710, USA.
Cryo-EM core, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, 27514, USA.
Nat Commun. 2022 Jul 1;13(1):3793. doi: 10.1038/s41467-022-31573-0.
How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.
细菌如何感知和响应氮水平是微生物生理学的核心问题。在革兰氏阳性菌中，氮稳态由编码谷氨酰胺合成酶（GS）的操纵子控制，GS 是一种十二聚体机器，可将铵同化到谷氨酰胺中，并与 GlnR 阻遏物结合。GlnR 通过结合受谷氨酰胺反馈抑制的 GS（FBI-GS）间接检测氮过量，从而激活其转录抑制功能。然而，这种调控回路的分子机制尚不清楚。在这里，我们描述了来自致病性和非致病性革兰氏阳性菌的 GS 和 FBI-GS-GlnR 复合物的生化和结构分析。这些结构表明，FBI-GS 结合 GS 的 GlnR C 末端结构域在其活性位点腔内，使两个 GlnR 单体并列，形成具有 DNA 结合能力的 GlnR 二聚体。FBI-GS-GlnR 相互作用稳定了无活性的 GS 构象。引人注目的是，这种相互作用还促进了某些 GS 中惊人的十二聚体到十四聚体的转变，打破了所有细菌 GS 都是十二聚体的范式。这些数据因此揭示了转录和酶调节的独特结构机制。
