Dipartimento di Scienze Biochimiche, "A Rossi Fanelli"- Sapienza Università di Roma, Italy.
Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Roma, Italy.
FEBS J. 2019 Nov;286(21):4245-4260. doi: 10.1111/febs.14959. Epub 2019 Jul 2.
The Small Ribosomal Subunit Biogenesis GTPase A (RsgA) is a bacterial assembly factor involved in the late stages of the 30S subunit maturation. It is a multidomain GTPase in which the central circularly permutated GTPase domain is flanked by an OB domain and a Zn-binding domain. All three domains participate in the interaction with the 30S particle thus ensuring an efficient coupling between catalytic activity and biological function. In vivo studies suggested the relevance of rsgA in bacterial growth and cellular viability, but other pleiotropic roles of RsgA are also emerging. Here, we report the 3D structure of RsgA from Pseudomonas aeruginosa (PaRsgA) in the GDP-bound form. We also report a biophysical and biochemical characterization of the protein in both the GDP-bound and its nucleotide-free form. In particular, we report a kinetic analysis of the RsgA binding to GTP and GDP. We found that PaRsgA is able to bind both nucleotides with submicromolar affinity. The higher affinity towards GDP (K = 0.011 μm) with respect to GTP (K = 0.16 μm) is mainly ascribed to a smaller GDP dissociation rate. Our results confirm that PaRsgA, like most other GTPases, has a weak intrinsic enzymatic activity (k = 0.058 min ). Finally, the biological role of RsgA in P. aeruginosa was investigated, allowing us to conclude that rsgA is dispensable for P. aeruginosa growth but important for drug resistance and virulence in an animal infection model. DATABASES: Coordinates and structure factors for the protein structure described in this manuscript have been deposited in the Protein Data Bank (https://www.rcsb.org) with the accession code 6H4D.
小核糖体亚基生物发生 GTP 酶 A（RsgA）是一种参与 30S 亚基成熟后期的细菌组装因子。它是一种多结构域 GTP 酶，其中中央循环排列的 GTP 酶结构域被一个 OB 结构域和一个 Zn 结合结构域包围。这三个结构域都参与与 30S 颗粒的相互作用，从而确保催化活性和生物学功能之间的有效偶联。体内研究表明 rsgA 与细菌生长和细胞活力有关，但 RsgA 的其他多效性作用也正在出现。在这里，我们报道了铜绿假单胞菌（PaRsgA）中 GDP 结合形式的 RsgA 的 3D 结构。我们还报告了该蛋白在 GDP 结合及其核苷酸游离形式下的生物物理和生化特性。特别是，我们报告了 RsgA 与 GTP 和 GDP 结合的动力学分析。我们发现 PaRsgA 能够以亚微摩尔亲和力结合两种核苷酸。与 GTP（K = 0.16μm）相比，对 GDP（K = 0.011μm）具有更高的亲和力主要归因于较小的 GDP 解离速率。我们的结果证实，像大多数其他 GTP 酶一样，PaRsgA 具有较弱的固有酶活性（k = 0.058min ）。最后，研究了 RsgA 在铜绿假单胞菌中的生物学作用，这使我们得出结论，rsgA 对于铜绿假单胞菌的生长不是必需的，但对于动物感染模型中的耐药性和毒力很重要。数据库：本文所述蛋白质结构的坐标和结构因子已被提交到蛋白质数据库（https://www.rcsb.org），登录号为 6H4D。
