Expression, purification, and crystallization of the RGS-like domain from the rho nucleotide exchange factor, PDZ-RhoGEF, using the surface entropy reduction approach

Lsc-homology domains are found in several eukaryotic nucleotide exchange fa ctors which act on Rho-family GTPases. They show limited amino acid sequenc e similarity to RGS proteins, which down-regulate the cellular signaling by the alpha -subunits of trimeric G-proteins and have been shown to interact with G alpha 12 and G alpha 13. It is believed that the RGS-like (RGSL) do main constitutes the functional link between G-protein-coupled receptors an d cytosolic Rho-GTPases. We report here the expression, purification, and c rystallization of the RGSL domain from the PDZ-RhoGEF. To obtain X-ray-grad e crystals we have used the recently proposed approach of crystallization b y mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target pro tein to reduce the cumulative conformational entropy of surface residues. O f the five mutants that were designed and prepared, the second one tried (K 463A, E465A, E466A) yielded crystals suitable for further analysis and diff racted X-rays to 2.8 Angstrom resolution on a home source. The crystals exh ibit hexagonal symmetry, space group P6(1)22 or P6(5)22, with unit cell par ameters a = b = 63.1 Angstrom, c = 202.1 Angstrom, and contain one molecule in the asymmetric unit. (C) 2001 Academic Press.