Structure of the RGS-like domain from PDZ-RhoGEF: Linking heterotrimeric Gprotein-coupled signaling to Rho GTPases

Background: The multidomain PDZ-RhoGEF is,one of many known guanine nucleot ide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related fa mily members play a critical role in a molecular signaling pathway from het erotrimeric G protein-coupled receptors to Rho proteins. A similar to 200 r esidue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with G alpha (12/13) proteins. To better unders tand structure-function relationships, we initiated crystallographic studie s of the RGSL domain from human PDZ-RhoGEF. Results: A recombinant construct of the RGSL domain was expressed in Escher ichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic a cid 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 Angstrom resolution, revealing an all-helical tertiary fold with the mutations located at inter molecular lattice contacts. Conclusions: The first nine helices adopt a fold similar to that observed f or RGS proteins, although the sequence identity with other such known struc tures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobi c interactions. Comparison with RGS proteins suggests features that are lik ely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable t o other proteins resistant to crystallization.