Fischer Martin, Frank Dominik, Rösler Reinhild, Johnsson Nils, Gronemeyer Thomas
Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany.
Core Unit Mass Spectrometry and Proteomics, Ulm University, Ulm, Germany.
Front Cell Dev Biol. 2022 Mar 3;10:771388. doi: 10.3389/fcell.2022.771388. eCollection 2022.
Septins are part of the cytoskeleton and polymerize into non-polar filaments of heteromeric hexamers or octamers. They belong to the class of P-loop GTPases but the roles of GTP binding and hydrolysis on filament formation and dynamics are not well understood. The basic human septin building block is the septin rod, a hetero-octamer composed of SEPT2, SEPT6, SEPT7, and SEPT9 with a stoichiometry of 2:2:2:2 (2-6-7-9-9-7-6-2). Septin rods polymerize by end-to-end and lateral joining into linear filaments and higher ordered structures such as rings, sheets, and gauzes. We purified a recombinant human septin octamer from for experimentation that is able to polymerize into filaments. We could show that the C-terminal region of the central SEPT9 subunit contributes to filament formation and that the human septin rod decreases the rate of actin polymerization. We provide further first kinetic data on the nucleotide uptake- and exchange properties of human hexameric and octameric septin rods. We could show that nucleotide uptake prior to hydrolysis is a dynamic process and that a bound nucleotide is exchangeable. However, the hydrolyzed γ-phosphate is not released from the native protein complex. We consequently propose that GTP hydrolysis in human septins does not follow the typical mechanism known from other small GTPases.
Septin蛋白是细胞骨架的一部分，能够聚合成异源六聚体或八聚体的非极性细丝。它们属于P环GTP酶家族，但GTP结合和水解在细丝形成及动力学方面的作用尚未完全明确。人类Septin蛋白的基本构建单元是Septin杆，它是一种异源八聚体，由SEPT2、SEPT6、SEPT7和SEPT9组成，化学计量比为2:2:2:2（2-6-7-9-9-7-6-2）。Septin杆通过端对端和侧向连接聚合成线性细丝以及更高阶的结构，如环、片层和网状结构。我们从……中纯化出一种重组人Septin八聚体用于实验，该八聚体能够聚合成细丝。我们发现中央SEPT9亚基的C末端区域有助于细丝形成，并且人Septin杆降低了肌动蛋白聚合的速率。我们提供了关于人六聚体和八聚体Septin杆核苷酸摄取和交换特性的进一步初步动力学数据。我们发现水解前的核苷酸摄取是一个动态过程，并且结合的核苷酸是可交换的。然而，水解产生的γ-磷酸并未从天然蛋白复合物中释放出来。因此，我们提出人类Septin蛋白中的GTP水解并不遵循其他小GTP酶已知的典型机制。
