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 factors which act on Rho-family GTPases. They show limited amino acid sequence 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 Galpha12 and Galpha13. It is believed that the RGS-like (RGSL) domain constitutes the functional link between G-protein-coupled receptors and cytosolic Rho-GTPases. We report here the expression, purification, and crystallization of the RGSL domain from the PDZ-RhoGEF. To obtain X-ray-grade crystals we have used the recently proposed approach of crystallization by mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target protein to reduce the cumulative conformational entropy of surface residues. Of the five mutants that were designed and prepared, the second one tried (K463A, E465A, E466A) yielded crystals suitable for further analysis and diffracted X-rays to 2.8 A resolution on a home source. The crystals exhibit hexagonal symmetry, space group P6(1) 22 or P6(5) 22, with unit cell parameters a = b = 63.1 A, c = 202.1 A, and contain one molecule in the asymmetric unit.
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22906-0011, USA.
S M
SM
Garrard
K L
KL
Longenecker
M E
ME
Lewis
P J
PJ
Sheffield
Z S
ZS
Derewenda
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D019277:Entropy; D004926:Escherichia coli; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry*; D064067:Rho Guanine Nucleotide Exchange Factors; D017386:Sequence Homology, Amino Acid
D020662:Guanine Nucleotide Exchange Factors; D020710:RGS Proteins; D064067:Rho Guanine Nucleotide Exchange Factors