Structural Biology Research Center, Institute of Material Structure Science, High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan.
J Biol Chem. 2010 Nov 19;285(47):36689-97. doi: 10.1074/jbc.M110.152132. Epub 2010 Sep 10.
Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.
许多 GTP 酶调节真核细胞内的运输和信号转导。鸟嘌呤核苷酸交换因子（GEF）通过催化 GDP 与 GTP 的交换来激活 GTP 酶。在这里，我们展示了与拟南芥 ARA7 的 GEF 反应的晶体学和生化研究，该蛋白是 Rab5 GTP 酶的植物同源物，与它的 GEF VPS9a 形成复合物，处于无核苷酸和 GDP 结合形式，以及与氨基膦酸 - 鸟苷酸酯和 ARA7·VPS9a(D185N)与 GDP 形成的复合物。在与 ARA7 形成复合物后，VPS9 楔入 ARA7 的开关 II 区域，抑制 Mg2+的协调，并降低 GDP 结合的稳定性。VPS9a 的天冬氨酸指直接识别 GDP β-磷酸，并将 ARA7 的 P 环赖氨酸从 GDP β-磷酸中拉向开关 II，进一步使 GDP 不稳定，以便在核苷酸交换反应中从 GDP 结合的中间物过渡到无核苷酸的中间物时释放 GDP。
