Stroupe C, Brunger A T
The Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305-548, USA.
J Mol Biol. 2000 Dec 8;304(4):585-98. doi: 10.1006/jmbi.2000.4236.
We have determined crystal structures of Sec4, a member of the Rab family in the G protein superfamily, in two states: bound to GDP, and to a non-hydrolyzable GTP analog, guanosine-5'-(beta, gamma)-imidotriphosphate (GppNHp). This represents the first structure of a Rab protein bound to GDP. Sec4 in both states grossly resembles other G proteins bound to GDP and GppNHp. In Sec4-GppNHp, structural features common to active Rab proteins are observed. In Sec4-GDP, the switch I region is highly disordered and displaced relative to the switch I region of Ras-GDP. In two of the four molecules of Sec4-GDP in the asymmetric unit of the Sec4-GDP crystals, the switch II region adopts a conformation similar to that seen in the structure of the small G protein Ran bound to GDP. This allows residues threonine 76, glutamate 80, and arginine 81 of Sec4 to make contacts with other conserved residues and water molecules important for nucleotide binding. In the other two molecules in the asymmetric unit, these interactions do not take place. This structural variability in both the switch I and switch II regions of GDP-bound Sec4 provides a possible explanation for the high off-rate of GDP bound to Sec4, and suggests a mechanism for regulation of the GTPase cycle of Rab proteins by GDI proteins.
我们已经确定了G蛋白超家族中Rab家族成员Sec4在两种状态下的晶体结构：与GDP结合以及与一种不可水解的GTP类似物鸟苷-5'-（β，γ）-亚氨基三磷酸（GppNHp）结合。这代表了Rab蛋白与GDP结合的首个结构。处于两种状态下的Sec4与其他结合GDP和GppNHp的G蛋白大体相似。在Sec4-GppNHp中，观察到了活性Rab蛋白共有的结构特征。在Sec4-GDP中，开关I区域高度无序，相对于Ras-GDP的开关I区域发生了位移。在Sec4-GDP晶体不对称单元中的四个Sec4-GDP分子中的两个中，开关II区域采用了一种与结合GDP的小G蛋白Ran结构中所见相似的构象。这使得Sec4的苏氨酸76、谷氨酸80和精氨酸81残基能够与其他对核苷酸结合很重要的保守残基和水分子形成接触。在不对称单元中的另外两个分子中，这些相互作用并未发生。结合GDP的Sec4的开关I和开关II区域的这种结构变异性为Sec4结合的GDP的高解离速率提供了一种可能的解释，并暗示了一种由GDI蛋白调节Rab蛋白GTP酶循环的机制。
