Splingard Anne, Ménétrey Julie, Perderiset Mylène, Cicolari Jérome, Regazzoni Karine, Hamoudi Fatima, Cabanié Lucien, El Marjou Ahmed, Wells Amber, Houdusse Anne, de Gunzburg Jean
Institut Curie, Centre de Recherche, Paris F-75248, France, INSERM U528, Paris F-75248, France.
J Biol Chem. 2007 Jan 19;282(3):1905-15. doi: 10.1074/jbc.M604363200. Epub 2006 Nov 15.
RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg.GDP at 2.1 A resolution. It reveals that the G-domain fold and Mg.GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an alpha-helical conformation that extends along the alpha5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.
RGK蛋白包括Rad、Gem、Rem1和Rem2，是Ras超家族中一个有趣的分支；它们的表达在转录水平受到调控，具有非典型的核苷酸结合基序，并且具有较大的N端和C端延伸。需要进行生化和结构研究以更好地了解此类蛋白的功能。在此，我们报道了RGK蛋白的首个结构：人Gem蛋白截短形式（G结构域加上C端延伸的第一部分）与Mg.GDP复合物的晶体结构，分辨率为2.1埃。结果表明，Gem的G结构域折叠和Mg.GDP结合位点与其他Ras家族GTP酶相似。C端延伸的第一部分采用α螺旋构象，沿着α5螺旋延伸并与开关区的末端相互作用。生化研究表明，与H-Ras相比，Gem对GDP和GTP的亲和力显著更低（微摩尔范围），与N端和C端延伸的有无无关，而其GTP酶活性高于H-Ras且受两个延伸部分的调节。我们展示了RGK蛋白开关II特有的庞大DXWEX基序如何影响开关I的构象和磷酸结合位点。总之，我们的数据表明Gem是一种真正的GTP酶，具有显著的结构和生化特征，这些特征应该会影响其调节和细胞活性。
