Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor.
Rho guanine-nucleotide exchange factors (RhoGEFs) activate Rho GTPases, and thereby regulate cytoskeletal structure, gene transcription, and cell migration. Leukemia-associated RhoGEF (LARG) belongs to a small subfamily of RhoGEFs that are RhoA-selective and directly activated by the Galpha12/13 family of heterotrimeric G proteins. Herein we describe the atomic structures of the catalytic Dbl homology (DH) and pleckstrin homology (PH) domains of LARG alone and in complex with RhoA. These structures demonstrate that the DH/PH domains of LARG can undergo a dramatic conformational change upon binding RhoA, wherein both the DH and PH domains directly engage RhoA. Through mutational analysis we show that full nucleotide exchange activity requires a novel N-terminal extension on the DH domain that is predicted to exist in a broader family of RhoGEFs that includes p115-RhoGEF, Lbc, Lfc, Net1, and Xpln, and identify regions within the LARG PH domain that contribute to its ability to facilitate nucleotide exchange in vitro. In crystals of the DH/PH-RhoA complex, the active site of RhoA adopts two distinct GDP-excluding conformations among the four unique complexes in the asymmetric unit. Similar changes were previously observed in structures of nucleotide-free Ras and Ef-Tu. A potential protein-docking site on the LARG PH domain is also evident and appears to be conserved throughout the Lbc subfamily of RhoGEFs.
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712-0165, USA.
Romana
R
Kristelly
Guang
G
Gao
John J G
JJ
Tesmer
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D010444:Peptide Elongation Factor Tu / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000502:physiology*
C576600:ARHGEF1 protein, human; C576601:ARHGEF3 protein, human; D020662:Guanine Nucleotide Exchange Factors; D009711:Nucleotides; D064067:Rho Guanine Nucleotide Exchange Factors; C066737:RHOA protein, human; D006153:Guanosine Diphosphate; D004247:DNA; D020558:GTP Phosphohydrolases; D010444:Peptide Elongation Factor Tu; D020742:rhoA GTP-Binding Protein