Institut de Biologie Structurale (IBS), Université Grenoble Alpes (UGA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), CNRS, F-38044 Grenoble, France.
Institut de Biologie Structurale (IBS), Université Grenoble Alpes (UGA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), CNRS, F-38044 Grenoble, France
Proc Natl Acad Sci U S A. 2021 Jan 12;118(2). doi: 10.1073/pnas.2014383118.
Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.
致病细菌和共生细菌通常需要抵抗宿主胃部的强酸环境。诱导型赖氨酸脱羧酶 LdcI 可缓冲细胞质和局部细胞外环境，以确保肠杆菌在低 pH 值下存活。在这里，我们通过生化和生物物理特性分析、负染和低温电子显微镜（cryo-EM）以及宽场和超分辨率荧光成像相结合，研究了 LdcI 的酸应激反应调控。由于荧光蛋白融合对天然 LdcI 十聚体具有有害影响，我们选择了用纳米体标记的内源性野生型 LdcI 在酸胁迫细胞中的三维定位，并表明它在细胞边缘形成明显的斑块。最近的假设认为，代谢酶在体内的聚集通常反映了它们作为刺激诱导调节的聚合形式，与这一假设一致，我们表明 LdcI 在生理相关的低 pH 值下体外组装成纤维。我们通过 cryo-EM 解决了这些纤维和在中性 pH 下形成的 LdcI 十聚体的结构，并通过突变分析证实了 LdcI 聚合的分子决定因素。最后，我们提出了 LdcI 在肠杆菌细胞内的功能模型，为进一步研究其超分子组织在酸应激反应中的作用提供了结构和机制基础。
