The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF.
The Dbl homology nucleotide exchange factors (GEFs) activate Rho family cytosolic GTPases in a variety of physiological and pathophysiological events. These signaling molecules typically act downstream of tyrosine kinase receptors and often facilitate nucleotide exchange on more than one member of the Rho GTPase superfamily. Three unique GEFs, i.e. p115, PDZ-RhoGEF, and LARG, are activated by the G-protein coupled receptors via the Galpha(12/13), and exhibit very selective activation of RhoA, although the mechanism by which this is accomplished is not fully understood. Based on the recently solved crystal structure of the DH-PH tandem of PDZ-RhoGEF in complex with RhoA (Derewenda, U., Oleksy, A., Stevenson, A. S., Korczynska, J., Dauter, Z., Somlyo, A. P., Otlewski, J., Somlyo, A. V., and Derewenda, Z. S. (2004) Structure (Lond.) 12, 1955-1965), we conducted extensive mutational and functional studies of the molecular basis of the RhoA selectivity in PDZ-RhoGEF. We show that while Trp(58) of RhoA is intimately involved in the interaction with the DH domain, it is not a selectivity determinant, and its interaction with PDZ-RhoGEF is unfavorable. The key selectivity determinants are dominated by polar contacts involving residues unique to RhoA. We find that selectivity for RhoA versus Cdc42 is defined by a small number of interactions.
Oleksy
Arkadiusz
A
Institute of Biochemistry and Molecular Biology, University of Wroclaw, 50-137 Wroclaw, Poland.
Opaliński
Łukasz
Ł
Derewenda
Urszula
U
Derewenda
Zygmunt S
ZS
Otlewski
Jacek
J
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D000939:Epitopes; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein