An in silico approach to determine inter-subunit affinities in human septin complexes

The septins are a conserved family of filament-forming guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. Correctly assembled septin structures are required for essential intracellular processes such as cytokinesis, vesicular transport, polarity establishment, and cellular adhesion. Septins structurally belong to the P-Loop NTPases but they do not mediate signals to effectors through GTP binding and hydrolysis. GTP binding and hydrolysis are believed to contribute to septin complex integrity, but biochemical approaches addressing this topic are hampered by the stability of septin complexes after recombinant expression and the lack of nucleotide-depleted complexes. To complement this limitation, we used a molecular dynamics based approach to determine inter-subunit free binding energies in available human septin dimer structures and in their apo forms, which we generated in silico. The nucleotide in the GTPase active subunits SEPT2 and SEPT7, but not in SEPT6, was identified as a stabilizing element in the G interface as it is coordinated at its ribose ring to conserved amino acids. Removal of GDP from SEPT2 and SEPT7 results in flipping of a conserved Arg residue and disruption of an extensive hydrogen bond network in the septin unique element, concomitant with a decreased inter-subunit affinity.

分隔蛋白（septins）是一类保守的、可形成丝状体的鸟苷酸结合蛋白，常被称作细胞骨架的第四组分。正确组装的分隔蛋白结构对于胞质分裂、囊泡运输、极性建立与细胞黏附等关键细胞内过程不可或缺。分隔蛋白在结构上归属于P环核苷三磷酸酶（P-Loop NTPases），但它们并不通过GTP结合与水解向效应蛋白传递信号。学界普遍认为GTP结合与水解有助于维持分隔蛋白复合物的结构完整性，但相关生化研究受限于两大瓶颈：一是重组表达获取的分隔蛋白复合物稳定性过强，难以开展针对性操控实验；二是缺乏核苷酸耗竭的分隔蛋白复合物。为弥补这一研究局限，我们采用基于分子动力学（molecular dynamics）的方法，对已解析的人类分隔蛋白二聚体结构，以及我们通过计算机模拟（in silico）生成的其脱辅基形式（apo forms），开展了亚基间自由结合能的测定。研究发现，GTP酶活性亚基SEPT2与SEPT7的G界面中，其结合的核苷酸可通过核糖环与保守氨基酸配位，成为稳定该界面的关键元件；而SEPT6则无此特征。将SEPT2与SEPT7中的GDP移除后，会导致保守精氨酸残基发生构象翻转，并破坏分隔蛋白特有元件内广泛存在的氢键网络，同时伴随亚基间亲和力的显著下降。