National Institute of Immunology, New Delhi, India.
Biophys J. 2010 Oct 6;99(7):2235-44. doi: 10.1016/j.bpj.2010.07.025.
The mechanism of oligomerization and its role in the regulation of activity in large GTPases are not clearly understood. Human guanylate binding proteins (hGBP-1 and 2) belonging to large GTPases have the unique feature of hydrolyzing GTP to a mixture of GDP and GMP with unequal ratios. Using a series of truncated and mutant proteins of hGBP-1, we identified a hydrophobic helix in the connecting region between the two domains that plays a critical role in dimerization and regulation of the GTPase activity. The fluorescence with 1-8-anilinonaphthalene sulfonate and circular dichroism measurements together suggest that in the absence of the substrate analog, the helix is masked inside the protein but becomes exposed through a substrate-induced conformational switch, and thus mediates dimerization. This is further supported by the intrinsic fluorescence experiment, where Leu(298) of this helix is replaced by a tryptophan. Remarkably, the enzyme exhibits differential GTPase activities depending on dimerization; a monomer produces only GDP, but a dimer gives both GDP and GMP with stimulation of the activity. An absolute dependence of GMP formation with dimerization demonstrates a cross talk between the monomers during the second hydrolysis. Similar to hGBP-1, hGBP-2 showed dimerization-related GTPase activity for GMP formation, indicating that this family of proteins follows a broadly similar mechanism for GTP hydrolysis.
寡聚化的机制及其在大 GTP 酶活性调节中的作用尚不清楚。属于大 GTP 酶的人类鸟苷酸结合蛋白（hGBP-1 和 2）具有独特的特性，可将 GTP 水解为 GDP 和 GMP 的混合物，且两者比例不等。我们使用 hGBP-1 的一系列截断和突变蛋白，鉴定出两个结构域之间连接区的一个疏水性螺旋，它在二聚化和 GTP 酶活性调节中起着关键作用。1-8-苯胺萘磺酸盐的荧光和圆二色性测量结果共同表明，在没有底物类似物的情况下，该螺旋被隐藏在蛋白质内部，但通过底物诱导的构象转变而暴露出来，从而介导二聚化。这进一步得到了本底荧光实验的支持，其中该螺旋的亮氨酸（Leu298）被色氨酸取代。值得注意的是，该酶根据二聚化表现出不同的 GTP 酶活性；单体仅产生 GDP，但二聚体在刺激活性的情况下同时产生 GDP 和 GMP。GMP 形成与二聚化的绝对依赖性表明，在第二次水解过程中单体之间存在串扰。类似于 hGBP-1，hGBP-2 表现出与 GMP 形成相关的 GTP 酶活性，表明该蛋白家族遵循广泛相似的 GTP 水解机制。
