Abstract
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.
Highlights
These proteins are thought to control the activity of L-type Ca2ϩ channels by interacting with their -subunit [2,3,4] as well as to intervene in cytoskeletal dynamics
Biochemistry and Structure of Gem properties of the regions of RGK proteins involved in interacting with nucleotides as well as the biochemical features of GDP/ GTP binding and GTP hydrolysis may be quite different from those of other Ras family proteins
We initially focused on the portion of Gem encompassing the G domain plus the first part of the C-terminal extension, devoid of the N-terminal extension, the CaM-binding region, and the seven C-terminal residues of the C-terminal extension (Gem⌬N⌬CaM, residues Met73–Arg264; see Fig. 1), to compare its biochemical and structural properties with those of H-Ras
Summary
CaM, calmodulin; TEV, tobacco etch virus; rTEV, recombinant His6-tagged TEV protease; GMP-PNP, guanylyl-5Ј-[,␥-imido]-triphosphate; GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); ␣Cter helix, C-terminal helix of Gem⌬N⌬CaM; GST, glutathione S-transferase. Biochemistry and Structure of Gem properties of the regions of RGK proteins involved in interacting with nucleotides as well as the biochemical features of GDP/ GTP binding and GTP hydrolysis may be quite different from those of other Ras family proteins. In order to investigate these questions, we have solved the crystal structure of a truncated form of human Gem, encompassing the G domain plus the first part of the C-terminal extension (Met73–Arg264) in complex with Mg1⁄7GDP at 2.1 Å resolution, and characterized its biochemical activities. We have investigated the influence of the N- and C-terminal extensions on the biochemical properties of Gem, including the association of CaM with its C-terminal region
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