Expression, purification, crystallization and preliminary structural characterization of the GTPase domain of human Rheb

Ras homologue enriched in brain (Rheb) represents a unique group of small GTPases and shares moderate sequence identity with the Ras/Rap subfamily. It acts downstream of nutrient signalling as the direct target of the tuberous sclerosis complex (TSC) and upstream of mTOR/S6K1/4EBP in the insulin-signalling pathway. The GTPase domain of human Rheb (hRheb) has been recombinantly expressed in Escherichia coli, purified and cocrystallized in complexes with GDP, GTP and GppNHp using the hanging-drop vapour-diffusion method. Crystals of the hRheb-GDP complex belong to space group P2₁2₁2 ₁, with unit-cell parameters a = 44.5, b = 52.3, c = 70.6 Å. The hRheb-GppNHp complex crystallized in two crystal forms: one has the same space group and unit-cell parameters as the hRheb-GDP complex and the other belongs to space group C222₁, with unit-cell parameters a = 102.9, b = 99.2, c = 48.0 Å. The hRheb-GTP complex also crystallized in two crystal forms: one belongs to space group C222₁, with unit-cell parameters a = 102.4, b = 98.3, c = 47.9 Å, and the other belongs to space group P2₁, with unit-cell parameters a = 77.3, b = 47.9, c = 71.9 Å, β = 89.0°. All these crystals diffract X-rays to better than 2.8 Å resolution and at least one diffraction data set has been collected for each crystal form using an in-house R-AXIS IV⁺⁺ diffractometer. Structural studies of hRheb in complexes with various substrates may provide insights into the recognition and specificity of substrate and the catalytic mechanism of mammalian Rhebs and shed light on the biological functions of Rhebs in the mTOR signalling pathway.