Sirajuddin Minhajuddin, Farkasovsky Marian, Zent Eldar, Wittinghofer Alfred
Abteilung Strukturelle Biologie, Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16592-7. doi: 10.1073/pnas.0902858106. Epub 2009 Sep 15.
Septins constitute a group of GTP-binding proteins involved in cytokinesis and other essential cellular functions. They form heterooligomeric complexes that polymerize into nonpolar filaments and are dynamic during different stages of the cell cycle. Posttranslational modifications and interacting partners are widely accepted regulators of septin filament function, but the contribution of nucleotide is undefined due to a lack of detailed structural information. Previous low-resolution structures showed that the G domain assembles into a linear polymer with 2 different interfaces involving the N and C termini and the G binding sites. Here we report the crystal structure of SEPT2 bound to GppNHp at 2.9 A resolution. GTP binding induces conformational changes in the switch regions at the G interfaces, which are transmitted to the N-terminal helix and also affect the NC interface. Biochemical studies and sequence alignment suggest that a threonine, which is conserved in certain subgroups of septins, is responsible for GTP hydrolysis. Although this threonine is not present in yeast CDC3 and CDC11, its mutation in CDC10 and CDC12 induces temperature sensitivity. Highly conserved contact residues identified in the G interface are shown to be necessary for Cdc3-10, but not Cdc11-12, heterodimer formation and cell growth in yeast. Based on our findings, we propose that GTP binding/hydrolysis and the nature of the nucleotide influence the stability of interfaces in heterooligomeric and polymeric septins and are required for proper septin filament assembly/disassembly. These data also offer a first rationale for subdividing human septins into different functional subgroups.
Septins是一组参与胞质分裂和其他重要细胞功能的GTP结合蛋白。它们形成异源寡聚复合物，聚合成非极性细丝，并且在细胞周期的不同阶段具有动态性。翻译后修饰和相互作用的伙伴是被广泛认可的septin细丝功能调节因子，但由于缺乏详细的结构信息，核苷酸的作用尚不清楚。先前的低分辨率结构表明，G结构域组装成线性聚合物，具有涉及N和C末端以及G结合位点的2个不同界面。在这里，我们报告了与GppNHp结合的SEPT2在2.9埃分辨率下的晶体结构。GTP结合诱导G界面处开关区域的构象变化，这些变化传递到N端螺旋，也影响NC界面。生化研究和序列比对表明，在septins的某些亚组中保守的苏氨酸负责GTP水解。虽然这种苏氨酸在酵母CDC3和CDC11中不存在，但其在CDC10和CDC12中的突变会诱导温度敏感性。在G界面中鉴定出的高度保守的接触残基被证明对于酵母中Cdc3 - 10异二聚体形成和细胞生长是必需的，但对于Cdc11 - 12则不是。基于我们的发现，我们提出GTP结合/水解和核苷酸的性质影响异源寡聚和聚合septins中界面的稳定性，并且是正确的septin细丝组装/拆卸所必需的。这些数据还为将人类septins细分为不同功能亚组提供了首个理论依据。
