
<ArticleTitle>Structure of the RGS-like domain from <prot>PDZ-RhoGEF</prot>: linking heterotrimeric g protein-coupled signaling to <prot>Rho</prot> GTPases.</ArticleTitle>
<AbstractText>BACKGROUND: The multidomain <prot>PDZ-RhoGEF</prot> is one of many known guanine nucleotide exchange factors that upregulate <prot>Rho</prot> GTPases. <prot>PDZ-RhoGEF</prot> and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to <prot>Rho</prot> proteins. A approximately 200 residue RGS-like (RGSL) domain in <prot>PDZ-RhoGEF</prot> and its homologs is responsible for the direct association with <prot>Galpha12</prot>/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human <prot>PDZ-RhoGEF</prot>. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.</AbstractText>
