Biochemical and structural characterization of the gem GTPase

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• RGK proteins (Rad, Gem, Rem1, Rem2) are a unique Ras superfamily branch.
• They have atypical nucleotide binding and large terminal extensions.
• Understanding RGK protein function requires biochemical and structural data.

Purpose of the Study:

• To determine the crystal structure of a human Gem protein fragment.
• To investigate the biochemical properties of Gem protein.
• To elucidate the structural basis for Gem's function and regulation.

Main Methods:

• X-ray crystallography to obtain the 2.1 A resolution structure of Gem (G domain + N-terminal extension) with Mg.GDP.
• Biochemical assays to measure GDP/GTP binding affinities and GTPase activity.
• Analysis of the DXWEX motif's structural impact.

Main Results:

• The Gem G-domain and Mg.GDP binding site resemble other Ras GTPases.
• The N-terminal extension forms an alpha-helix interacting with the interswitch.
• Gem exhibits lower GDP/GTP affinities but higher GTPase activity than H-Ras, regulated by extensions.
• The DXWEX motif alters switch I and phosphate-binding site conformation.

Conclusions:

• Gem is a bona fide GTPase with distinctive structural and biochemical characteristics.
• These features likely impact Gem's regulatory mechanisms and cellular roles.
• The study provides a structural basis for understanding RGK protein function.