Structural insights into Rab21 GTPase activation mechanism by molecular dynamics simulations

Rab proteins belong to the family of monomeric GTPases which are involved in the cellular membrane trafficking. Rab21 protein exists in interchangeable GTP- and GDP-bound states. Rabs switch between two active and inactive conformations like other GTPases. The inactive form of Rab is bound to GDP while its active form is bounded with the GTP. Interexchange between active and inactive form is mediated by the GDP/GTP exchange factor (GEF) which catalyses the conversion from GDP-bound to GTP-bound form, thereby activating the Rab. While the GTP hydrolysis of Rabs is regulated by a GTPase-activating protein (GAP) which causes Rab inactivation. Here, we report the structural flexibility of the Rab21-GTP and Rab21-GDP complexes by docking and molecular dynamics (MD) simulations. Structural analysis of exchange mechanisms of the co-factors complexed with Rab21 reveals that Cys29, Thr33, His48, Gln78 and Lys133 are essentially important in the activation of proteins. Furthermore, a significant change in the orientation of the interacting co-factors, with slight variation in the free energy of binding was observed. Complexation of GEF with Rab21-GTP and Rab21-GDP reveal a flipping of the switchable residues. Finally, 50 ns MD simulations confirm that the GTP-bound Rab21 complex is thermodynamically more favoured than the corresponding GDP-bound complex. This study provides a detailed understanding of the structural elements involved in the conformational changes of Rab21.

Rab蛋白属于单体GTP酶（monomeric GTPases）家族，参与细胞内膜运输（cellular membrane trafficking）过程。Rab21蛋白存在可相互转换的GTP结合态与GDP结合态。与其他GTP酶类似，Rabs蛋白会在活性与非活性两种构象之间切换：Rab的非活性形式与GDP结合，而活性形式则与GTP结合。活性与非活性形式之间的相互转换由GDP/GTP交换因子（GDP/GTP exchange factor, GEF）介导，该因子可催化GDP结合态向GTP结合态的转化，从而激活Rab蛋白。而Rabs蛋白的GTP水解过程则由GTP酶激活蛋白（GTPase-activating protein, GAP）调控，该蛋白会引发Rab蛋白的失活。本研究通过分子对接与分子动力学（molecular dynamics, MD）模拟，解析了Rab21-GTP复合物与Rab21-GDP复合物的结构柔性。对与Rab21结合的辅助因子的交换机制进行结构分析后发现，Cys29、Thr33、His48、Gln78与Lys133对于蛋白的激活具有至关重要的作用。此外，研究还观察到相互作用的辅助因子的取向发生了显著变化，而其结合自由能仅存在微小波动。GEF与Rab21-GTP、Rab21-GDP形成的复合物结构显示，可切换残基发生了翻转。最后，50纳秒的分子动力学模拟证实，GTP结合态的Rab21复合物在热力学上比相应的GDP结合态复合物更为稳定。本研究深入阐明了Rab21构象变化所涉及的结构元件。